Plant cyclopropane fatty acid synthase genes, proteins, and uses thereof

ABSTRACT

The present invention provides cyclopropane fatty acid synthase genes and proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also provides methods of using cyclopropane fatty acid synthase genes and proteins, including in their expression in transgenic organisms and in the production of cyclopropane fatty acids in plant oils, and in particular seed oils.

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/345,152 and 60/393,937, filed on Dec. 21, 2001 and Jul. 3,2002, respectively, both pending.

FIELD OF THE INVENTION

The present invention relates to isolated cyclopropane fatty acidsynthase genes and polypeptides. The present invention also providesmethods for using cyclopropane fatty acid synthase genes, polypeptides,and synthase products.

BACKGROUND OF TIIE INVENTION

Vegetable oils are utilized not only in the food industry, but alsoincreasingly in the chemical industry. The utility of any particular oildepends upon chemical and physico-chemical properties of the oil, whichis determined by the composition of the constituent fatty acids. Plantoils are often modified to meet industrial specifications. Suchmodification of vegetable oil has typically been achieved by chemicalmeans (fractionation, interesterification, hydrogenation, or otherchemical derivatization), but genetic means (plant breeding, mutagenesisand genetic engineering) are increasingly being used to provide noveloil feedstocks.

One class of particular interest is the class of fatty acids containingthree carbon carbocyclic rings, which includes the cyclopropane fattyacids (CPA-FAs) and cyclopropene fatty acids (CPE-FAs). The cyclopropenering confers two unique properties for these fatty acids and oils.First, hydrogenation produces large amounts of methyl-branched fattyacids. These will give the low temperature properties equivalent tounsaturated fatty acids and their esters without the oxidativesusceptibility of the double bonds, and therefore may find uses inlubrication and related fields (Kai, Y. (1982) J. Am. Oil Chem. Soc. 59:300-305). Moreover, the methyl-branched fatty acids may also be viewedas a replacement for isostearic acids formed in dimer acid production,and isostearic is an article of commerce in the oleochemical industrywhere it is used in applications as diverse as cosmetics and lubricantadditives. Second, the cyclopropene ring is highly strained and readilyring opens in an exothermic reaction with electrophiles. Oils with highlevels of cyclopropene fatty acid, such as Sterculia foetida oil,self-polymerize at elevated temperatures. This property is particularlyapplicable to the production of coatings and polymers. In the Sterculiafoetida oil, sterculic acid reacts with acetic acid to produce a varietyof acetyl esters, as well as with short or medium chain saturated fattyacids to yield monounsaturated estolide products (Kircher (1964) J. OrgChem. 29:1979-1982); all of these products can be further hydrogenatedand saponified or hydrolyzed to form hydroxy fatty acids. Reaction ofthe oil with dibasic carboxylic acids should result in polymers.Moreover, sterculic acid might also be used as a biocide in fatty acidsoap formulation.

On the other hand, CPE-FAs are considered an anti-nutritional factor infood oils. Many seed lipids containing CPE-FAs are extensively consumedby humans, especially in tropical areas (Ralaimanarivo et al. (1982)Lipids 17 (1): 1-10). It is well documented that dietary CPE-FAs lead tothe accumulation of hard fats and other physiological disorders inanimals (Phelps et al. (1965) Poultry Science 44: 358-394; Page et al.(1997) Comparative Biochemistry And Physiology B-Biochemistry &Molecular Biology 118 (1): 79-84). CPE-FAs are strong inhibitors ofvariety of desaturases in animals (Cao et al (1993) Biochimica etBiophysica Acta 1210 (1): 27-34; Fabrias et al. (1996) Journal of LipidResearch 37 (7): 1503-1509; Fogerty et al. (1972) Lipids 7(5): 335-338),which might be the cause of at least some of the observed disorders.Because of these health concerns, vegetable oils containing CPE-FAs mustbe treated with high temperature or hydrogenation before consumption.These treatments add to the oil processing costs, and also result in thepresence of a certain percentage of trans fatty acids produced due thehydrogenation; the presence of such trans fatty acids are alsoundesirable. Therefore, it would be desirable to obtain plant oils withgreatly reduced levels of CPE-FAs, as the availability of such oilswould significantly reduce the processing costs, decrease the presenceof undesirable hydrogenated fatty acids, and enhance the value of theoils for food consumption. Elimination of CPE-FAs would also enhance thevalue of unprocessed seeds or seed meal, such as cottonseed, as animalfeed.

Currently, there are no commercial sources of oils rich in CPE-FAs. Itis believed that plant CPE-FAs are synthesized from CPA-FAs viadesaturation. E. coli and other bacteria have the ability to synthesizefatty acids containing a cyclopropane ring. The reaction is catalyzed bythe enzyme cyclopropane fatty acid synthase (also known as cyclopropanesynthase or unsaturated phospholipid methyltransferase; E.C. 2.1.1.16)and involves the addition of a methylene group from S-adenosylmethionineacross the double bond of phospholipid hexadecenoyl or octadecenoylgroups. CPA-FAs (CFAs), such as dihydrosterculate (DHS) arecharacterized by a saturated 3-membered ring, as shown by the followingstructure, where X═OH for a free fatty acid, or an alcohol moiety for anester:

The cyclopropane fatty acid synthase gene in E. coli has been cloned andsequenced (Grogan et al. (1997) J. Bacteriol. 158:286-295 and Wang etal. (1992) Biochemistry 31: 11020-11028). No CPE-FAs have been reportedin bacteria.

CPA-FAs (CPA-FA) and CPE-FAs (CPE-FA) are not widely distributed in highplants, but they are found in the seed oils of limited families,including the Malvaceae, Sterculiaceae, Bombaceae, Tilaceae, Mimosaceaeand Sapindaceae (Smith (1970) Progress in the Chemistry of Fats andOther Lipids (Pergamon Press: New York) Vol. 11, pp139-177; Christie(1970) in Topics in Lipid Chemistry (Gunstone F D Ed.; Logos Press:London) Vol. 1, pp1-49; Badami and Patil (1981) Prog. Lipid Res. 19:119-153). The CPA-FAs and CPE-FAs are not confined to seeds. Kuiper andStuiver ((1972) Plant Physiol. 49: 307-309) have described long-chainCPA-FAs in various polar lipid classes of leaves of early spring plants.Yano et al. ((1972) Lipids 7: 30-34) and Schmid and Patterson ((1988)Phytochem. 27: 2831-2834) report that CPA-FAs and CPE-FAs are found inroot, leaf stem and callus tissue in plants of the Malvaceae.

In a few plant species, CPA-FAs can reach high levels, in other words upto 40% in Litchi chinensis (Vickery et al. (1980) J. Am. Oil Chem. Soc.57: 87-91; and Gaydou et al. (1993) J. Ag. Food Chem. 41: 886-890).However, it is more common to find CPE-FAs, particularly in the orderMalvales (for example, as in the report by Bohannon and Kleiman (1978)Lipids 13: 270-273), and a biosynthetic pathway of CPE-FAs throughCPA-FAs was postulated by Yano et al. ((1972) Lipids 7: 35-45). Thus, inplants, CPE-FAs exist primarily in the form of sterculic and malvalicacids, where malvalic acid is the one carbon homolog of sterculic acidand is obtained by chain shortening at the carboxyl end by ∀-oxidation.The CPE-FAs are usually accompanied with small amount of correspondingCPA-FAs, dihydrosterculic and dihydromalvalic acids. However, there havebeen no confirmed identified and isolated plant genes which encodeproteins which are capable of synthesizing CPA-FAs. Moreover, plantswith high levels of cyclopropene are not grown commercially.

Therefore, it would be desirable to be able to generate vegetable oilswith high amounts of cyclopropane and CPE-FAs. One route is byidentifying and isolating a plant gene which is capable of synthesizingCPA-FAs. Such a gene could then be used to transform oil crop plants.Identification of such a gene could also be used to reduce the levels ofCPE-FAs by gene silencing techniques.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising cyclopropanefatty acid synthase (“CPA-FAS”) genes and polypeptides. The presentinvention is not limited to any particular nucleic acid or amino acidsequence. The present invention also provides methods for using CPA-FASgenes and polypeptides.

Accordingly, in some embodiments, the present invention providescompositions comprising an isolated nucleic acid sequence encoding aplant CPA-FAS or portions thereof. In some embodiments, the plant isfrom the order Malvales; in some further embodiments, the plant is aSterculia plant or a cotton plant. In some particular embodiments, thenucleic acid sequence encodes a Sterculia CPA-FAS or a portion thereof;in some further particular embodiments, the Sterculia is Sterculiafoetida, and in even further particular embodiments, the isolatednucleic acid sequence comprises SEQ ID NO: 1. In other particularembodiments, the nucleic acid sequence encodes a cotton CPA-FAS or aportion thereof; in further particular embodiments, the cotton isGossypium arboreum, and in even further particular embodiments, theisolated nucleic acid sequence comprises at least one of SEQ ID NOs:3,4, 5, or 6. In other embodiments, the present invention provides anisolated nucleic acid sequence comprising a plant cyclopropane fattyacid synthase gene. In other embodiments, the present invention providescompositions comprising an isolated nucleic acid sequence comprising anucleic acid sequence encoding an amino terminus of a plant cyclopropanefatty acid synthase; in some particular embodiments, the amino terminusof a plant cyclopropane fatty acid synthase comprises approximately thefirst about 420 to about 470 amino acids of a plant cyclopropane fattyacid synthase; in other particular embodiments, the amino terminus of aplant cyclopropane fatty acid synthase comprises the first approximately440 amino acids of SEQ ID NO:2 or the amino acid sequence of SEQ IDNO:7; and in yet other particular embodiments, the coding sequencecomprises about the first 1410 nucleotides of SEQ ID NO: 1 or comprisesSEQ ID NO:3. In other embodiments, the present invention providescompositions comprising an isolated nucleic acid sequence comprising anucleic acid sequence encoding an amino acid sequence homologous to anamino terminus of a plant cyclopropane fatty acid synthase; in someparticular embodiments, the amino terminus of a plant cyclopropane fattyacid synthase comprises approximately the first about 420 to about 470amino acids of a plant cyclopropane fatty acid synthase; in otherparticular embodiments, the amino terminus of a plant cyclopropane fattyacid synthase comprises the first approximately 440 amino acids of SEQID NO:2 or the amino acid sequence of SEQ ID NO:7.

The present invention is not limited to nucleic acid sequences encodinga plant CPA-FAS; indeed, it is contemplated that the present inventionencompasses isolated nucleic acid sequences encoding homologs, variants,and portions or fragments of a plant CPA-FAS. Accordingly, in someembodiments, the present invention provides compositions comprising anisolated nucleic acid sequence that hybridizes under conditions of lowto high stringency to a nucleic sequence comprising SEQ ID NOs: 1, 3, 4,5, or 6. In some particular embodiments, the isolated nucleic acidhybridizes under conditions of high stringency to a nucleic sequencecomprising SEQ ID NOs: 1, 3, 4, 5, or 6. In further embodiments, thehybridizing sequence encodes a polypeptide that catalyzes the additionof a methylene group across an unsaturated center of an unsaturatedfatty acid. In other embodiments, the isolated nucleic acid sequenceshybridize under conditions of low to high stringency to a nucleicsequence comprising about the first 1410 nucleotides of SEQ ID NO: 1 orcomprising SEQ ID NO:3. In some particular embodiments, the isolatednucleic acid hybridizes under conditions of high stringency to a nucleicsequence comprising about the first 1410 nucleotides of SEQ ID NO:1 orcomprising SEQ ID NO:3. In further embodiments, the hybridizing sequenceencodes a polypeptide that catalyzes the addition of a methylene groupacross an unsaturated center of an unsaturated fatty acid.

In other embodiments, the present invention provides compositionscomprising an isolated nucleic acid sequence encoding a plant CPA-FAS,wherein the synthase competes for binding to an unsaturated fatty acidsubstrate with a protein encoded by a nucleic acid sequence comprisingSEQ ID NOs: 1 or at least one of SEQ ID NOs:3, 4, 5, or 6; in otherembodiments, the present invention provides compositions comprising anisolated nucleic acid sequence encoding a plant CPA-FAS, wherein thesynthase competes for binding to an unsaturated fatty acid substratewith a protein comprising amino acid sequence SEQ ID NO:2 or at leastone of SEQ ID NO:7, 8, 9, or 10.

In other embodiments, the present invention provides compositionscomprising an antisense sequence corresponding to any of the nucleicacid sequences encoding a plant CPA-FAS as described above. In yet otherembodiments, the present invention provides compositions comprisingribozymes and hairpin loops targeted to any of the plant CPA-FAS codingsequences described above; in further embodiments, the present inventionprovides compositions comprising a nucleic acid sequence encodingribozymes and hairpin loops targeted to any of the plant CPA-FAS codingsequences described above. In yet other embodiments, the presentinvention provides compositions comprising siRNAs targeted to a sequencein an mRNA transcribed from any of the nucleic acid sequences encoding aplant cyclopropane fatty acid synthase as described above; in yetfurther embodiments, the present invention provides compositionscomprising nucleic acid sequences encoding an siRNA targeted to asequence in an mRNA transcribed from any of the nucleic acid sequencesencoding a plant cyclopropane fatty acid synthase as described above.

In some embodiments of the present invention, a nucleic acid sequencedescribed above is operably linked to a heterologous promoter. Infurther embodiments, the sequence described above linked to aheterologous promoter is contained within a vector.

In other embodiments, the invention provides compositions comprising apurified polypeptide encoded by any of the nucleic acid sequencesdescribed above which when transcribed and translated result inexpression of a polypeptide; in some embodiments, the polypeptide ispurified from a recombinant organism transformed with any of the nucleicsequences described above which when transcribed and translated resultin expression of a polypeptide. In other embodiments, the presentinvention provides compositions comprising a purified plant CPA-FAS orportions thereof. In some embodiments, the plant CPA-FAS is purifiedfrom a plant of order Malvales; in other embodiments, the plant CPA-FASis purified from Sterculia; in other embodiments, the CPA-FAS ispurified from Sterculia foetida; in yet other embodiments, the plantCPA-FAS comprises amino acid sequence SEQ ID NO:2. In other embodiments,the plant CPA-FAS is purified from cotton; in yet other embodiments, theplant CPA-FAS comprises at least one of amino acid SEQ ID NOs:7, 8, 9,or 10.

In further embodiments, the present invention provides an organismtransformed with any of the nucleic acid sequences described above. Inother embodiments, the present invention provides an organismtransformed with a heterologous gene encoding a plant CPA-FAS or aportion thereof; in some embodiments, the gene is from a Malvales plant;in other embodiments the gene is a Sterculia gene; in yet otherembodiments, the gene is a Sterculia foetida gene; and in yet otherembodiments, the gene encodes SEQ ID NO:2. In yet other embodiments, thegene is a cotton gene; in other particular embodiments, the gene encodesa CPA-FAS comprising at least one of amino acid SEQ ID NOs: 7, 8, 9, or10.

In other embodiments, the present invention provides organismsco-transformed with a first heterologous gene encoding a plant CPA-FASand with a second heterologous gene encoding a fatty acid desaturase. Inyet other embodiments, the present invention provides organismsco-transformed with a heterologous gene encoding a fusion polypeptidecomprising a plant CPA-FAS and a fatty acid desaturase.

In other embodiments of the present invention, a transformed organism asdescribed above is either a plant or microorganism. In a preferredembodiment, the organism is a plant. In other embodiments, a plant cellis transformed with any of the nucleic acid sequences described above.In yet other embodiments, a plant seed is transformed with any of thenucleic acid sequences described above. In yet other embodiments, theinvention provides oils from plants transformed as described above. Infurther embodiments, a transformed organism as described above is abacteria; in other embodiments, the invention provides oils from suchtransformed bacteria.

In other embodiments, the present provides methods for expressing aplant cyclopropane fatty acid synthase in a plant, comprising providinga vector comprising any of the nucleic acid sequences described abovewhich encode a plant CPA-FAS or portion thereof and plant tissue, andtransfecting the plant tissue with the vector under conditions such thatthe synthase is expressed. In other embodiments, the present inventionprovides methods for decreasing expression of CPA-FAS in plants,comprising providing a vector comprising a nucleic acid sequenceencoding an antisense sequence corresponding to any of the nucleic acidsequences described above which encode a plant CPA-FAS or portionthereof, and plant tissue, and transfecting the plant tissue with thevector under conditions such that the antisense sequence is expressedand the expression of CPA-FAS is decreased. In yet other embodiments,the present invention provides methods for decreasing expression ofCPA-FAS in plants, comprising providing a vector encoding an siRNAtargeted to any of the nucleic acid sequences described above whichencode a plant CPA-FAS or portion thereof, and plant tissue, andtransfecting the plant tissue with the vector under conditions such thatthe siRNA is expressed and the expression of CPA-FAS is decreased.

In further embodiments, the invention provides methods for producing avariant of plant CPA-FAS, comprising providing a nucleic acid sequenceencoding a plant CPA-FAS, and mutagenizing the nucleic acid sequence soas to produce a variant; in still other embodiments, the methods furthercomprise screening the variant for activity. In some embodiments, thevariant is a fragment of CPA-FAS which has CPA-FAS activity. In otherembodiments, the variant is a mutated CPA-FAS which has altered CPA-FASactivity when compared to the unmutated CPA-FAS.

In other embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such as fattycyclopropane fatty acids, in vitro, comprising providing a purifiedplant CPA-FAS and at least one fatty acid substrate of the enzyme, andincubating the synthase with the substrate under conditions such thatfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, are produced.

In particular embodiments, the substrate of the plant CPA-FAS is anunsaturated fatty acid with variable chain length and from none to oneor more additional functional groups comprising acetylenic bonds,conjugated acetylenic and ethylenic bonds, allenic groups, cyclopenteneand furan rings, or epoxy-, hydroxy- and keto-groups, wherein thesubstrates are either free fatty acids, or fatty acids incorporated intoa larger molecule; in other particular embodiments, the substrate isoleic or palmitoleic acid.

In other embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such ascyclopropane fatty acids, in vitro, comprising providing an isolatednucleic sequence encoding a plant CPA-FAS and at least one fatty acidsubstrate of the enzyme, and incubating the sequence with the substratein a transcription/translation system under conditions such that thesequence is expressed and cyclopropane fatty acids are produced. Inparticular embodiments, the substrate of the plant CPA-FAS is selectedfrom the group as described above.

In other embodiments, the invention provides methods of producing fattyacids with three carbon carbocyclic rings, such as cyclopropane fattyacids, in vitro, comprising providing a purified plant CPA-FAS, apurified fatty acid desaturase and at least one fatty acid substrate ofthe desaturase, and incubating the CPA-FAS and the desaturase with thesubstrate(s) under conditions such that fatty acids with three carboncarbocyclic rings, such as cyclopropane fatty acids, are produced. Inpreferred embodiments, the fatty acid substrate(s) of the desaturase isany fatty acid to which an additional double bond can be added, whichdouble bond can be the site of addition of a methylene group by theCPA-FAS.

In further embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such ascyclopropane fatty acids, by fermentation, comprising providing amicroorganism transformed with a heterologous gene encoding a plantCPA-FAS and at least one substrate of the plant CPA-FAS, and incubatingthe microorganism with the substrate under conditions such that fattyacids with three carbon carbocyclic rings, such as cyclopropane fattyacids, are produced. In particular embodiments, the fatty acid substrateof the CPA-FAS is a member of the group as described above.

In other embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such ascyclopropane fatty acids, by fermentation, comprising providing amicroorganism co-transformed with a first heterologous gene encoding aplant CPA-FAS and with a second heterologous gene encoding a fatty aciddesaturase and at least one substrate of the desaturase, and incubatingthe microorganism with the substrate under conditions such that fattyacids with three carbon carbocyclic rings, such as cyclopropane fattyacids, are produced. In preferred embodiments, the fatty acid substrateof the desaturase is a member of the group described above.

In further embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such ascyclopropane fatty acids, by fermentation, comprising providing amicroorganism transformed with a heterologous gene encoding a fusionpolypeptide comprising a plant CPA-FAS and a fatty acid desaturase andat least one substrate of the desaturase, and incubating themicroorganism with the substrate under conditions such that fatty acidswith three carbon carbocyclic rings, such as cyclopropane fatty acids,are produced. In particular embodiments, the fatty acid substrate of thedesaturase is a member of the group described above.

In other embodiments, the present invention provides methods ofproducing fatty acids with three carbon carbocyclic rings, such ascyclopropane fatty acids, in a plant comprising providing a plant and aheterologous gene encoding a plant CPA-FAS, and transforming the plantwith the heterologous gene such that fatty acids with three carboncarbocyclic rings, such as cyclopropane fatty acids, are produced. Inother embodiments, the present invention provides methods of producingfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, in a plant comprising growing a plant transformed with aheterologous gene encoding a plant CPA-FAS under conditions such thatfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, are produced. In yet other embodiments, the presentinvention provides methods of producing fatty acids with three carboncarbocyclic rings, such as cyclopropane fatty acids, in a plantcomprising providing a plant, a first heterologous gene encoding a plantCPA-FAS, and a second heterologous gene encoding a desaturase, andco-transforming the plant with the first heterologous gene and with thesecond heterologous gene such that fatty acids with three carboncarbocyclic rings, such as cyclopropane fatty acids, are produced. Inother embodiments, the present invention provides methods of producingfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, in a plant comprising growing a plant transformed with afirst heterologous gene encoding a plant CPA-FAS, and with a secondheterologous gene encoding a desaturase, under conditions such thatfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, are produced. In still other embodiments, the presentinvention provides methods of producing fatty acids with three carboncarbocyclic rings, such as cyclopropane fatty acids, in a plantcomprising providing a plant and a heterologous gene encoding a fusionpolypeptide comprising a plant CPA-FAS and a fatty acid desaturase, andtransforming a plant with the heterologous gene, such that fatty acidswith three carbon carbocyclic rings, such as cyclopropane fatty acids,are produced. In other embodiments, the present invention providesmethods of producing fatty acids with three carbon carbocyclic rings,such as cyclopropane fatty acids, in a plant comprising growing a plantwith a heterologous gene encoding a fusion polypeptide comprising aplant CPA-FAS and a fatty acid desaturase under conditions such thatfatty acids with three carbon carbocyclic rings, such as cyclopropanefatty acids, are produced.

In other particular embodiments, the present invention providestransgenic plants comprising any of the nucleic acid sequences of theinvention described above, where the nucleic acid sequences are undercontrol of promoters that control expression of the nucleic acidsequence in a target tissue of the plant or in a target developmentalphase of the plant, or under control of promoters that are inducible. Itis contemplated that such transgenic plants may be used for any of themethods described above for producing cyclopropane fatty acids inplants.

In yet other embodiments, the present invention provides methods forscreening plant CPA-FASs comprising providing a candidate plant CPA-FASand analyzing said candidate CPA-FAS for the presence of the S-adenosylmethionine binding motif and catalytic cysteine in the amino acidsequence; preferably, the motif and catalytic cysteine are present inthe carboxy-terminus of the plant CPA-FAS.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the proposed pathway for the biosynthesis of sterculicacid.

FIG. 2 shows the profile of fatty acid accumulation during Sterculiaseed development.

FIG. 3 shows the distribution of Sterculia seed ESTs along the putativeSterculia cyclopropane synthase cDNA derived from clones R50D5 andR15C3. The start codon is located at 98 bp and the stop codon is locateat 2690 bp. In each box, the first number is the number of ESTs, and thesecond number is the location where the EST(s), starts. The arrowindicates the direction and region covered by the given EST(s).

FIG. 4 shows a nucleic acid sequence (SEQ ID NO: 1) for Sterculiacyclopropane fatty acid synthase (SCPA-FAS).

FIG. 5 shows the deduced amino acid sequence (SEQ ID NO:2) for theSterculia cyclopropane fatty acid synthase (SCPA-FAS) shown in FIG. 4.

FIG. 6 shows the GC/MS analysis of FAMEs from yeast grown for 48 hr inmedium supplemented with 200 mg/L oleic acid. Panel (A) shows the totalion chromatogram of FAMEs from control transgenic yeast containing theconstruct pYES2/CT-LacZ. Panel (B) shows the total ion chromatogram ofFAMEs from yeast containing the construct comprising a coding sequencefor Sterculia cyclopropane synthase, pYES2/CT-SCPAS. An additional peakwith the retention time of 34.44 min can be seen in this chromatogram.Panel (C) shows the mass spectrum of the unique additional peak observedin the chromatogram of FAMES from panel (B). The mass spectrum is thesame as that of the dihydrosterculic acid standard (see FIG. 8(A)). Theinsert is the enlarged image containing the additional peak.

FIG. 7 shows the total ion chromatograms of FAMEs from transgenictobacco cells. Panel (A) shows the saturated FAMEs from controltransgenic tobacco cells transformed with the empty construct pE1776.Panel (B) shows the saturated FAMEs from transgenic tobacco cellstransformed with the construct containing a coding sequence forSterculia cyclopropane synthase, pE1776-SCPAS. A major additional peakwith a retention time of 35.69 min can be seen in this sample. Panel (C)shows the dihydrosterculic acid methyl-ester standard with a retentiontime of 35.69 min.

FIG. 8 shows a comparison of the mass spectra of the dihydrosterculicacid standard and the additional peak found in transgenic cellstransformed with a coding sequence for Sterculia cyclopropane synthase.Panel (A) shows the total ion chromatogram of the dihydrosterculic acidmethyl-ester standard. Panel (B) shows the mass spectrum of thedihydrosterculic acid methyl-ester standard. Several ions are unique todihydrosterculic acid methyl-ester, including the molecular ionsm/z=310, M-32 is 278, and M-74 is 236. Panel (C) shows the total ionchromatogram of saturated FAMEs from transgenic tobacco cellstransformed with a construct containing the coding sequence for theSterculia cyclopropane synthase, pE1776-SCPAS. Panel (D) shows the massspectrum of the peak with the retention time of 35.69 min seen in panel(C). The mass spectrum of this peak is nearly identical to that of thedihydrosterculic acid methyl-ester standard, with a signature ofmolecular ions 310, M-32 of 278, and M-74 of 236.

FIG. 9 shows the dihydrosterculic acid content of 15 independenttransgenic tobacco lines transformed with a coding sequence forSterculia cyclopropane synthase.

FIG. 10 Panel (A) shows an amino acid alignment of the carboxy terminalportion of the Sterculia cyclopropane synthase with other cyclopropanesynthases and cyclopropane synthase-like enzymes. The — line indicatesthe S-adenosyl-methionine binding site, and the * is the catalyticimportant Cysteine. Panel (B) shows the phylogenetic relationship amongthese enzymes.

FIG. 11 shows an amino acid alignment between Sterculia and bacterialcyclopropane fatty acid synthase. The portion from about amino acid 470to amino acid 864 (the conserved carboxyl terminus) was used to prepareantibody to the Sterculia enzyme.

FIG. 12 shows the locations of the primers relative to the Sterculiacyclopropane fatty acid synthase coding sequence (the primers are shownabove the coding sequence), where the primers were used for PCRamplification of the coding sequence for the conserved carboxylterminus.

FIG. 13 shows the protein expressed in BL21 (SEQ ID NO:11). Thehighlighted portion is derived from the vector, which contains a6-histidine tag for purification.

FIG. 14 shows nucleic acid sequences of three ESTs discovered in cotton(Gossypium arboreum) by blasting NCBI database with the amino acidsequence from Sterculia cyclopropane fatty acid synthase. Panel A showsEST1 (SEQ ID NO:3), panel B shows EST2 (SEQ ID NO:4), and panel C showsEST3 (SEQ ID NO:5).

FIG. 15 shows results of a contig analysis of the three cotton ESTsshown in FIG. 14, which demonstrates that EST2 and 3 are overlap ESTs.Panel A shows the results graphically; panel B shows a nucleic acidsequence alignment of EST2, EST3, and Contig 1, and panel C shows thenucleic acid sequence of Contig 1 (SEQ ID NO:6).

FIG. 16 shows predicted amino acid sequences for Sterculia cyclopropanefatty acid synthase (panel A, SEQ ID NO:2), cotton EST1 (panel B, SEQ IDNO:7), cotton EST2 (panel C, SEQ ID NO:8), cotton EST3 (panel D, SEQ IDNO:9) and cotton Contig 1 (EST2_(—)3, panel E, SEQ ID NO:10).

FIG. 17 shows an alignment of the amino acid sequence of the Sterculiacyclopropane fatty acid synthase (SEQ ID NO:2), and the predicted aminoacid sequences of two cotton EST sequences, EST1 (SEQ ID NO:7) and theContig 1 (EST2_(—)3, SEQ ID NO:10).

FIG. 18 shows a Western blot analysis of different tissues (embryos,leaves, stems, and roots) from cotton and of embryo tissue fromSterculia with an antibody against Sterculia cyclopropane fatty acidsynthase (CPSA-FAS).

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to compositions comprising plantcyclopropane fatty acid (CPA-FAS) genes and polypeptides. The presentinvention encompasses compositions comprising both native andrecombinant forms of the enzyme, as well as mutant and variant forms,some of which possess altered characteristics relative to the wild-type.The present invention also provides methods for using plant CPA-FASgenes, polypeptides, and synthase products.

In some embodiments, the present invention provides novel isolatednucleic acid sequences encoding a plant cyclopropane fatty acidsynthase. In other embodiments, the invention provides isolated nucleicacid sequences encoding mutants, variants, homologs, chimeras, andfusions of plant cyclopropane fatty acid synthase. In other embodiments,the present invention provides methods of generating such sequences. Inother embodiments, the present invention provides methods of cloning andexpressing such sequences, as well as methods of purifying and assayingthe expression product of such sequences.

In additional embodiments, the invention provides purified plant CPA-FASpolypeptides. In other embodiments, the present invention providesmutants, variants, homologs, chimeras, and fusion proteins of plantCPA-FAS. In some embodiments, the present invention provides methods ofpurifying, and assaying the biochemical activity of wild type as well asmutants, variants, homologs, chimeras, and fusions of plant cyclopropanefatty acid synthase polypeptides, as well as methods of generatingantibodies to such proteins.

In some embodiments, the present invention provides methods of usingnovel isolated nucleic acid sequences encoding a plant cyclopropanefatty acid synthase to produce products of the synthase activity. Insome embodiments, the methods involve adding the sequences to in vitrotranscription and translations systems which include the substrates ofthe synthase, such that the products of the synthase may be recovered.In other embodiments, the methods involve transforming organisms withthe sequences such that the sequences are expressed and products of thesynthase are produced. In particular embodiments, the products arerecovered. In other embodiments, the products remain in situ.

In some embodiments, the present invention provides methods of usingrecombinant plant CPA-FAS polypeptides to produce products of thesynthase activity. In some embodiments, the methods involve adding thepolypeptides to in vitro systems which include the substrates of thesynthase, such that the products of the synthase may be recovered.

In other embodiments, the methods involve transforming a plant with anovel isolated nucleic acid sequence encoding a plant cyclopropane fattyacid synthase such that products of the synthase are produced.

In some embodiments, the present invention provides an organismtransformed with heterologous gene encoding a plant cyclopropane fattyacid synthase. In some embodiments, the organism is a microorganism. Inother embodiments, the organism is a plant.

In some embodiments, the present invention also provides a celltransformed with an heterologous gene encoding a plant cyclopropanefatty acid synthase. In some embodiments, the cell is a microorganism.In other embodiments, the cell is a plant cell.

In other embodiments, the present invention provides a plant seedtransformed with a nucleic acid sequence encoding a plant cyclopropanefatty acid synthase. In yet other embodiments, the present inventionprovides an oil from a plant transformed with a plant cyclopropane fattyacid synthase.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases as used herein are defined below:

The term “plant” is used in it broadest sense. It includes, but is notlimited to, any species of woody, ornamental or decorative, crop orcereal, fruit or vegetable plant, and photosynthetic green algae (forexample, Chlamydomonas reinhardtii). It also refers to a plurality ofplant cells which are largely differentiated into a structure that ispresent at any stage of a plant's development. Such structures include,but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.The term “plant tissue” includes differentiated and undifferentiatedtissues of plants including those present in roots, shoots, leaves,pollen, seeds and tumors, as well as cells in culture (for example,single cells, protoplasts, embryos, callus, etc.). Plant tissue may bein planta, in organ culture, tissue culture, or cell culture. The term“plant part” as used herein refers to a plant structure or a planttissue.

The term “crop” or “crop plant” is used in its broadest sense. The termincludes, but is not limited to, any species of plant or algae edible byhumans or used as a feed for animals or used, or consumed by humans, orany plant or algae used in industry or commerce.

The term “oil-producing species” refers to plant species which produceand store triacylglycerol in specific organs, primarily in seeds. Suchspecies include but are not limited to soybean (Glycine max), rapeseedand canola (including Brassica napus and B. campestris), sunflower(Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa(Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeisguineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum),castor (Ricinus communis) and peanut (Arachis hypogaea). The group alsoincludes non-agronomic species which are useful in developingappropriate expression vectors such as tobacco, rapid cycling Brassicaspecies, and Arabidopsis thaliana, and wild species which may be asource of unique fatty acids.

The term “Sterculia” refers to a plant or plants from the genusSterculia of the family Sterculiaceae of the order Malvales.Non-limiting examples of Sterculia include plants from the species S.foetida, S. oblongata, S. Rinopetala, S. urens, S. villosa. The termalso refers to S. foetida plants from which nucleic acid sequence SEQ IDNO: 1 was isolated.

The term “cotton” refers to a cotton plant or plants from the genusGossypium of the family Malvaceae of the order Malvales; the genusGossypium contains at least 39 species. Non-limiting examples of cottoninclude plants from the species G. arboreum, G. hirsutum, G. herbaceum,and G. barbadense. The term also refers to Gossypium arboreum plantsfrom which nucleic acid sequences SEQ ID NOs: 3, 4, and 6 were derived.

The term plant cell “compartments or organelles” is used in its broadestsense. The term includes but is not limited to, the endoplasmicreticulum, Golgi apparatus, trans Golgi network, plastids, sarcoplasmicreticulum, glyoxysomes, mitochondrial, chloroplast, and nuclearmembranes, and the like.

The term “host cell” refers to any cell capable of replicating and/ortranscribing and/or translating a heterologous gene.

The term “methylene-added fatty acid” refers to a fatty acid where amethylene group has been added to the hydrocarbon chain to producemethyl- or methylene-branched or three-member carbocyclic ringstructure.

The term “three member carbocyclic ring fatty acid” refers to a fattyacid with a cyclopropane or cyclopropene ring.

The term “cyclopropane fatty acid” (CPA-FA) refers to a fatty acidcharacterized by a saturated 3-membered ring. The term “cyclopropenefatty acid” (CPE-FA) refers to a fatty acid characterized by anunsaturated 3-membered ring.

The term “cyclopropane fatty acid synthase” (CPA-FAS) or “cyclopropanesynthase” refers to a polypeptide with the capacity to synthesize afatty acid containing a cyclopropane ring. When presented withsubstrates other than cis-monounsaturated fatty acids, or aftermodification of just a few amino acids, the polypeptide is contemplatedto function more broadly as a three-member carbocyclic ring fatty acidsynthase or as a methylene-added fatty acid synthase. The basic reactioninvolves addition of a methylene group across the double bond. Thus, thepolypeptide catalyzes the addition of a methylene group across theunsaturated center of an unsaturated fatty acid, and includes theaddition of a methylene group across a double bond of an acyl group. Themethionine can be obtained from S-adenosylmethionine. Typically, theacyl group is a hexadecenoyl or octadecenoyl group, although other fattyacyl groups of different chain lengths and degrees of unsaturation arealso contemplated as substrates. The fatty acyl group is believed to beesterified to a phospholipid; such phospholipid substrates includephosphatidylcholine, phosphatidylethanolamine, and phospatidylglycerol.The term “recombinant cyclopropane fatty acid synthase” (recombinantCPA-FAS) refers to the expression product of a recombinant nucleic acidmolecule or of a heterologous gene encoding CPA-FAS. Having “CPA-FASactivity” refers to having the functionality of a CPA-FAS, or having thecapacity to synthesize a fatty acid containing a cyclopropane ring orcatalyze the reaction described above. A “plant CPA-FAS” is an enzymeoriginally obtained from a plant source; the enzyme may be modifiedwhere such modifications include but are not limited to truncation,amino acid deletions, additions, and substitutions, and glycosylation,and where the resulting modified enzyme possesses CPA-FAS activity.

The term “competes for binding” is used in reference to a firstpolypeptide with enzymatic activity which binds to the same substrate asdoes a second polypeptide with enzymatic activity, where the secondpolypeptide is variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (for example, kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides.

The term “chimera” when used in reference to a polypeptide refers to theexpression product of two or more coding sequences obtained fromdifferent genes, that have been cloned together and that, aftertranslation, act as a single polypeptide sequence. Chimeric polypeptidesare also referred to as “hybrid” polypeptides. The coding sequencesincludes those obtained from the same or from different species oforganisms.

The term “fusion” when used in reference to a polypeptide refers to achimeric protein containing a protein of interest joined to an exogenousprotein fragment (the fusion partner). The fusion partner may servevarious functions, including enhancement of solubility of thepolypeptide of interest, as well as providing an “affinity tag” to allowpurification of the recombinant fusion polypeptide from a host cell orfrom a supernatant or from both. If desired, the fusion partner may beremoved from the protein of interest after or during purification.

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablegreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (for example, replacement of leucinewith isoleucine). More rarely, a variant may have “non-conservative”changes (for example, replacement of a glycine with a tryptophan).Similar minor variations may also include amino acid deletions orinsertions (in other words additions), or both. Guidance in determiningwhich and how many amino acid residues may be substituted, inserted ordeleted without abolishing biological activity may be found usingcomputer programs well known in the art, for example, DNAStar software.Variants can be tested in functional assays. Preferred variants haveless than 10%, and preferably less than 5%, and still more preferablyless than 2% changes (whether substitutions, deletions, and so on).

The term “gene” refers to a nucleic acid (for example, DNA or RNA)sequence that comprises coding sequences necessary for the production ofan RNA, or a polypeptide or its precursor (for example, proinsulin). Afunctional polypeptide can be encoded by a full length coding sequenceor by any portion of the coding sequence as long as the desired activityor functional properties (for example, enzymatic activity, ligandbinding, signal transduction, etc.) of the polypeptide are retained. Theterm “portion” when used in reference to a gene refers to fragments ofthat gene. The fragments may range in size from a few nucleotides to theentire gene sequence minus one nucleotide. Thus, “a nucleotidecomprising at least a portion of a gene” may comprise fragments of thegene or the entire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript The mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “heterologous gene” refers to a gene encoding a factor that isnot in its natural environment (in other words, has been altered by thehand of man). For example, a heterologous gene includes a gene from onespecies introduced into another species. A heterologous gene alsoincludes a gene native to an organism that has been altered in some way(for example, mutated, added in multiple copies, linked to a non-nativepromoter or enhancer sequence, etc.). Heterologous genes may compriseplant gene sequences that comprise cDNA forms of a plant gene; the cDNAsequences may be expressed in either a sense (to produce mRNA) oranti-sense orientation (to produce an anti-sense RNA transcript that iscomplementary to the mRNA transcript). Heterologous genes aredistinguished from endogenous plant genes in that the heterologous genesequences are typically joined to nucleotide sequences comprisingregulatory elements such as promoters that are not found naturallyassociated with the gene for the protein encoded by the heterologousgene or with plant gene sequences in the chromosome, or are associatedwith portions of the chromosome not found in nature (for example, genesexpressed in loci where the gene is not normally expressed).

The term “oligonucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

The term “an oligonucleotide having a nucleotide sequence encoding agene” or “a nucleic acid sequence encoding” a specified polypeptiderefers to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a gene productThe coding region may be present in either a cDNA, genomic DNA or RNAform. When present in a DNA form, the oligonucleotide may besingle-stranded (in other words, the sense strand) or double-stranded.Suitable control elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The terms “complementary” and “complementarity” refer to polynucleotides(in other words, a sequence of nucleotides) related by the base-pairingrules. For example, for the sequence “A-G-T,” is complementary to thesequence “T-C-A.” Complementarity may be “partial,” in which only someof the nucleic acids' bases are matched according to the base pairingrules. Or, there may be “complete” or “total” complementarity betweenthe nucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (in other words, identity). “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those nucleotide residues thatare identical and in the same relative positions in their respectivelarger sequences. Calculations of identity may be performed byalgorithms contained within computer programs such as “GAP” (GeneticsComputer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). Apartially complementary sequence is one that at least partially inhibits(or competes with) a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (in other words, the hybridization) of a sequence which iscompletely homologous to a target under conditions of low stringency.This is not to say that conditions of low stringency are such thatnon-specific binding is permitted; low stringency conditions requirethat the binding of two sequences to one another be a specific (in otherwords, selective) interaction. The absence of non-specific binding maybe tested by the use of a second target which lacks even a partialdegree of complementarity (for example, less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄XH₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5× Denhardt's reagent [50× Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 :g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42EC when a probe of about 500 nucleotides in lengthis employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄XH₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 :g/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (for example, increasing the temperatureof the hybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(in other words, it is the complement of) the single-stranded nucleicacid sequence under conditions of low to high stringency as describedabove.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (in other words,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementary between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids. A single moleculethat contains pairing of complementary nucleic acids within itsstructure is said to be “self-hybridized.”

The term “T_(m)” refers to the “melting temperature” of a nucleic acid.The melting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the T_(m) of nucleic acidsis well known in the art. As indicated by standard references, a simpleestimate of the T_(m) value may be calculated by the equation:T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (Se for example, Anderson and Young, Quantitative FilterHybridization (1985) in Nucleic Acid Hybridization). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” refers to the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.With “high stringency” conditions, nucleic acid base pairing will occuronly between nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (in other words, replication that is template-dependent butnot dependent on a specific template). Template specificity is heredistinguished from fidelity of replication (in other words, synthesis ofthe proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Q ∃replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al. (1972) Proc. Natl. Acad. Sci. USA, 69:3038).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlin et al(1970) Nature, 228:227). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace (1989) Genomics,4:560). Finally, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor prim erhybridization with the target sequences and not hybridization withnon-target sequences (H. A. Erlich (ed.) (1989) PCR Technology, StocktonPress).

The term “amplifiable nucleic acid” refers to nucleic acids that may beamplified by any amplification method. It is contemplated that“ampliflable nucleic acid” will usually comprise “sample template.”

The term “sample template” refers to nucleic acid originating from asample that is analyzed for the presence of “target” (defined below). Incontrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (in otherwords, in the presence of nucleotides and an inducing agent such as DNApolymerase and at a suitable temperature and pH). The primer ispreferably single stranded for maximum efficiency in amplification, butmay alternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (in other words, denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (for example, hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.

The terms “PCR product,” “PCR fragment,” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

The term “amplification reagents” refers to those reagents(deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid 15, template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

The term “reverse-transcriptase” or “RT-PCR” refers to a type of PCRwhere the starting material is mRNA. The starting mRNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a “template” for a “PCR” reaction.

The term “gene expression” refers to the process of converting geneticinformation encoded in a gene into RNA (for example, mRNA, rRNA, tRNA,or snRNA) through “transcription” of the gene (in other words, via theenzymatic action of an RNA polymerase), and into protein, through“translation” of mRNA. Gene expression can be regulated at many stagesin the process. “Up-regulation” or “activation” refers to regulationthat increases the production of gene expression products (for example,RNA or protein), while “down-regulation” or “repression” refers toregulation that decrease production. Molecules (for example,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “regulatory element” refers to a genetic element which controlssome aspect of the expression of nucleic acid sequences. For example, apromoter is a regulatory element which facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements are splicing signals, polyadenylation signals, terminationsignals, etc.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237, 1987). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect, mammalian and plant cells.Promoter and enhancer elements have also been isolated from viruses andanalogous control elements, such as promoters, are also found inprokaryotes. The selection of a particular promoter and enhancer dependson the cell type used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review, see Voss,et al., Trends Biochem. Sci., 11:287, 1986; and Maniatis, et al., supra1987).

The terms “promoter element,” “promoter,” or “promoter sequence” as usedherein, refer to a DNA sequence that is located at the 5′ end (in otherwords precedes) the protein coding region of a DNA polymer. The locationof most promoters known in nature precedes the transcribed region. Thepromoter functions as a switch, activating the expression of a gene. Ifthe gene is activated, it is said to be transcribed, or participating intranscription. Transcription involves the synthesis of mRNA from thegene. The promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA.

Promoters may be tissue specific or cell specific. The term “tissuespecific” as it applies to a promoter refers to a promoter that iscapable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue (for example, seeds) in therelative absence of expression of the same nucleotide sequence ofinterest in a different type of tissue (for example, leaves). Tissuespecificity of a promoter may be evaluated by, for example, operablylinking a reporter gene to the promoter sequence to generate a reporterconstruct, introducing the reporter construct into the genome of a plantsuch that the reporter construct is integrated into every tissue of theresulting transgenic plant, and detecting the expression of the reportergene (for example, detecting mRNA, protein, or the activity of a proteinencoded by the reporter gene) in different tissues of the transgenicplant. The detection of a greater level of expression of the reportergene in one or more tissues relative to the level of expression of thereporter gene in other tissues shows that the promoter is specific forthe tissues in which greater levels of expression are detected. The term“cell type specific” as applied to a promoter refers to a promoter whichis capable of directing selective expression of a nucleotide sequence ofinterest in a specific type of cell in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of cell within the same tissue. The term “cell type specific” whenapplied to a promoter also means a promoter capable of promotingselective expression of a nucleotide sequence of interest in a regionwithin a single tissue. Cell type specificity of a promoter may beassessed using methods well known in the art, for example,immunohistochemical staining. Briefly, tissue sections are embedded inparaffin, and paraffin sections are reacted with a primary antibodywhich is specific for the polypeptide product encoded by the nucleotidesequence of interest whose expression is controlled by the promoter. Alabeled (for example, peroxidase conjugated) secondary antibody which isspecific for the primary antibody is allowed to bind to the sectionedtissue and specific binding detected (for example, with avidin/biotin)by microscopy.

Promoters may be constitutive or regulatable. The term “constitutive”when made in reference to a promoter means that the promoter is capableof directing transcription of an operably linked nucleic acid sequencein the absence of a stimulus (for example, heat shock, chemicals, light,etc.). Typically, constitutive promoters are capable of directingexpression of a transgene in substantially any cell and any tissue.Exemplary constitutive plant promoters include, but are not limited toSD Cauliflower Mosaic Virus (CaMV SD; see for example, U.S. Pat. No.5,352,605, incorporated herein by reference), mannopine synthase,octopine synthase (ocs), superpromoter (see for example, WO 95/14098),and ubi3 (see for example, Garbarino and Belknap (1994) Plant Mol. Biol.24:119-127) promoters. Such promoters have been used successfully todirect the expression of heterologous nucleic acid sequences intransformed plant tissue.

In contrast, a “regulatable” promoter is one which is capable ofdirecting a level of transcription of an operably linked nuclei acidsequence in the presence of a stimulus (for example, heat shock,chemicals, light, etc.) which is different from the level oftranscription of the operably linked nucleic acid sequence in theabsence of the stimulus.

The enhancer and/or promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (in otherwords, molecular biological techniques) such that transcription of thegene is directed by the linked enhancer or promoter. For example, anendogenous promoter in operable combination with a first gene can beisolated, removed, and placed in operable combination with a secondgene, thereby making it a “heterologous promoter” in operablecombination with the second gene. A variety of such combinations arecontemplated (for example, the first and second genes can be from thesame species, or from different species.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamHI/BclI restriction fragment anddirects both termination and polyadenylation (Sambrook, supra, at16.6-16.7).

The term “selectable marker” refers to a gene which encodes an enzymehaving an activity that confers resistance to an antibiotic or drug uponthe cell in which the selectable marker is expressed, or which confersexpression of a trait which can be detected for example, luminescence orfluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotransferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

The term “vector refers to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

The terms “expression vector” or “expression cassette” refer to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

The term “transfection” refers to the introduction of foreign DNA intocells. Transfection may be accomplished by a variety of means known tothe art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,glass beads, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, viral infection, biolistics (forexample, particle bombardment) and the like.

The terms “infecting” and “infection” when used with a bacterium referto co-incubation of a target biological sample, (for example, cell,tissue, etc.) with the bacterium under conditions such that nucleic acidsequences contained within the bacterium are introduced into one or morecells of the target biological sample.

The term “Agrobacterium” refers to a soil-borne, Gram-negative,rod-shaped phytopathogenic bacterium which causes crown gall. The term“Agrobacteriuti” includes, but is not limited to, the strainsAgrobacterium tumefaciens, (which typically causes crown gall ininfected plants), and Agrobacterium rhizogens (which causes hairy rootdisease in infected host plants). Infection of a plant cell withAgrobacterium generally results in the production of opines (forexample, nopaline, agropine, octopine etc.) by the infected cell. Thus,Agrobacterium strains which cause production of nopaline (for example,strain LBA4301, C58, A208, GV3101) are referred to as “nopaline-type”Agrobacteria; Agrobacterium strains which cause production of octopine(for example, strain LBA4404, Ach5, B6) are referred to as“octopine-type” Agrobacteria; and Agrobacterium strains which causeproduction of agropine (for example, strain EHA105, EHA101, A281) arereferred to as “agropine-type” Agrobacteria.

The terms “bombarding, “bombardment,” and “biolistic bombardment” referto the process of accelerating particles towards a target biologicalsample (for example, cell, tissue, etc.) to effect wounding of the cellmembrane of a cell in the target biological sample and/or entry of theparticles into the target biological sample. Methods for biolisticbombardment are known in the art (for example, U.S. Pat. No. 5,584,807,the contents of which are incorporated herein by reference), and arecommercially available (for example, the helium gas-drivenmicroprojectile accelerator (PDS-1000/He, BioRad).

The term “microwounding” when made in reference to plant tissue refersto the introduction of microscopic wounds in that tissue. Microwoundingmay be achieved by, for example, particle bombardment as describedherein.

The term “transgenic” when used in reference to a plant or fruit or seed(in other words, a “transgenic plant” or “transgenic fruit” or a“transgenic seed”) refers to a plant or fruit or seed that contains atleast one heterologous gene in one or more of its cells. The term“transgenic plant material” refers broadly to a plant, a plantstructure, a plant tissue, a plant seed or a plant cell that contains atleast one heterologous gene in one or more of its cells.

The terms “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in, the originallytransformed cell are included in the definition of transformants.

The term “wild-type” when made in reference to a gene refers to a genewhich has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product which has the characteristics of a geneproduct isolated from a naturally occurring source. A wild-type gene isthat which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” when made in reference to agene or to a gene product refers, respectively, to a gene or to a geneproduct which displays modifications in sequence and/or functionalproperties (in other words, altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

The term “antisense” refers to a deoxyribonucleotide sequence whosesequence of deoxyribonucleotide residues is in reverse 5′ to 3′orientation in relation to the sequence of deoxyribonucleotide residuesin a sense strand of a DNA duplex. A “sense strand” of a DNA duplexrefers to a strand in a DNA duplex which is transcribed by a cell in itsnatural state into a “sense mRNA.” Thus an “antisense” sequence is asequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene by interfering with theprocessing, transport and/or translation of its primary transcript ormRNA. The complementarity of an antisense RNA may be with any part ofthe specific gene transcript, in other words, at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence. Inaddition, as used herein, antisense RNA may contain regions of ribozymesequences that increase the efficacy of antisense RNA to block geneexpression. “Ribozyme” refers to a catalytic RNA and includessequence-specific endoribonucleases. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of preventing theexpression of the target protein.

The term “siRNAs” refers to short interfering RNAs. In some embodiments,siRNAs comprise a duplex, or double-stranded region, of about 18-25nucleotides long; often siRNAs contain from about two to four unpairednucleotides at the 3′ end of each strand. At least one strand of theduplex or double-stranded region of a siRNA is substantially homologousto or substantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense strand;” thestrand homologous to the target RNA molecule is the “sense strand,” andis also complementary to the siRNA antisense strand. siRNAs may alsocontain additional sequences; non-limiting examples of such sequencesinclude linking sequences, or loops, which link the two strands of thedouble strand, as well as stem and other folded structures, which may bepresent within the linking sequence. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “target RNA molecule” refers to an RNA molecule to which atleast one strand of the short double-stranded region of an siRNA ishomologous or complementary. Typically, when such homology orcomplementary is about 100%, the siRNA is able to silence or inhibitexpression of the target RNA molecule. Although it is believed thatprocessed mRNA is a target of siRNA, the present invention is notlimited to any particular hypothesis, and such hypotheses are notnecessary to practice the present invention. Thus, it is contemplatedthat other RNA molecules may also be targets of siRNA. Such targetsinclude unprocessed mRNA, ribosomal RNA, and viral RNA genomes.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector which is not integrated into thegenome. The expression of the gene is either completely or partiallyinhibited. RNAi may also be considered to inhibit the function of atarget RNA; the function of the target RNA may be complete or partial.

The term “posttranscriptional gene silencing” or “PTGS” refers tosilencing of gene expression in plants after transcription, and appearsto involve the specific degradation of mRNAs synthesized from generepeats.

The term “overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. The term “cosuppression” refers to theexpression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. The term “altered levels” refers to theproduction of gene product(s) in transgenic organisms in amounts orproportions that differ from that of normal or non-transformedorganisms.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “Southern blot analysis” and “Southern blot” and “Southern”refer to the analysis of DNA on agarose or acrylamide gels in which DNAis separated or fragmented according to size followed by transfer of theDNA from the gel to a solid support, such as nitrocellulose or a nylonmembrane. The immobilized DNA is then exposed to a labeled probe todetect DNA species complementary to the probe used. The DNA may becleaved with restriction enzymes prior to electrophoresis. Followingelectrophoresis, the DNA may be partially depurinated and denaturedprior to or during transfer to the solid support. Southern blots are astandard tool of molecular biologists (J. Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp9.31-9.58).

The term “Northern blot analysis” and “Northern blot” and “Northern” asused herein refer to the analysis of RNA by electrophoresis of RNA onagarose gels to fractionate the RNA according to size followed bytransfer of the RNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized RNA is then probedwith a labeled probe to detect RNA species complementary to the probeused. Northern blots are a standard tool of molecular biologists (J.Sambrook, et al. (1989) supra, pp 7.39-7.52).

The terms “Western blot analysis” and “Western blot” and “Western”refers to the analysis of protein(s) (or polypeptides) immobilized ontoa support such as nitrocellulose or a membrane. A mixture comprising atleast one protein is first separated on an acrylamide gel, and theseparated proteins are then transferred from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized proteins areexposed to at least one antibody with reactivity against at least oneantigen of interest. The bound antibodies may be detected by variousmethods, including the use of radiolabeled antibodies.

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably.

As used herein, where “amino acid sequence” is recited herein to referto an amino acid sequence of a protein molecule, “amino acid sequence”and like terms, such as “polypeptide” or “protein” are not meant tolimit the amino acid sequence to the complete, native amino acidsequence associated with the recited protein molecule; furthermore, an“amino acid sequence” can be deduced from the nucleic acid sequenceencoding the protein.

The term “portion” when used in reference to a protein (as in “a portionof a given protein”) refers to fragments of that protein. The fragmentsmay range in size from four amino acid residues to the entire aminosequence minus one amino acid.

The term “homology” when used in relation to amino acids refers to adegree of similarity or identity. There may be partial homology orcomplete homology (in other words, identity). “Sequence identity” refersto a measure of relatedness between two or more proteins, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those amino acid residues thatare identical and in the same relative positions in their respectivelarger sequences. Calculations of identity may be performed byalgorithms contained within computer programs.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature. Forexample, a given DNA sequence (for example, a gene) is found on the hostcell chromosome in proximity to neighboring genes; RNA sequences, suchas a specific mRNA sequence encoding a specific protein, are found inthe cell as a mixture with numerous other mRNA s which encode amultitude of proteins. However, isolated nucleic acid encoding a plantCPA-FAS includes, by way of example, such nucleic acid in cellsordinarily expressing a DES, where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide will contain at a minimum the sense or coding strand (inother words, the oligonucleotide may single-stranded), but may containboth the sense and anti-sense strands (in other words, theoligonucleotide may be double-stranded).

The term “purified” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated. An “isolated nucleic acid sequence” is therefore a purifiednucleic acid sequence. “Substantially purified” molecules are at least60% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated. Theterm “purified” or “to purify” also refer to the removal of contaminantsfrom a sample. The removal of contaminating proteins results in anincrease in the percent of polypeptide of interest in the sample. Inanother example, recombinant polypeptides are expressed in plant,bacterial, yeast, or mammalian host cells and the polypeptides arepurified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

The term “sample” is used in its broadest sense. In one sense it canrefer to a plant cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The presently claimed invention provides compositions comprisingisolated plant CPA-FAS genes and polypeptides, and in particular tocompositions comprising isolated Sterculia and cotton CPA-FAS genes andpolypeptides. The present invention also provides methods for usingplant CPA-FAS genes, polypeptides, and synthase products; such methodsinclude but are not limited to plant CPA-FAS genes and polypeptides inthe production of cyclopropane fatty acids. The description belowprovides specific, but not limiting, illustrative examples ofembodiments of the present invention.

I. Plant Cyclopropane Fatty Acid Synthase Genes and Polypeptides

The biosynthetic pathway of CPA-FAs in bacteria is well characterized(Grogan and Cronan (1997) Microbiol Molecular Biol Rev 61 (4): 429441).The first cyclopropane synthase gene was cloned from E. coli, based uponits ability to complement CPA-FA deficient mutant (Grogan and Cronan(1984) J Bacteriol 158 (1): 286-295). It was clearly demonstrated thatbacterial CPA-FAs were directly synthesized from mono-unsaturated fattyacids by addition of a methylene group, derived fromS-adenosylmethionine, cross the double bond. However, the substrates ofthe enzyme appear to be mono-unsaturated fatty acids esterified tophospholipids, most likely phosphatidylethanolamine (Grogan and Cronan(1997) Microbiol Molecular Biol Rev 61 (4): 429-441). After theidentification of bacterial cyclopropane synthase, three genes from Mtuberculosis were found with the ability to introduce a cyclopropanering onto mycolic acids (Yuan and Barry (1996) Proc Nat'l Acad Sci USA93 (23): 12828-12833; Yuan et al. (1995) Proc Nat'l Acad Sci USA 92(14): 6630-6634).

Even though the existence of CPE-FAs in plants has been known for atleast several decades, the biosynthesis of this particular kind of fattyacids has received very little attention to date. In attempt tounderstand the biosynthesis of CPE-FAs, Yano et al ((1972) Lipids 7:35-45) conducted in vivo labeling experiments with several species ofMalvaceae. The authors concluded that the pathway of CPE-FA synthesisinvolved an initial formation of dihydrosterculic acid from oleic acidand a subsequent desaturation of dihydrosterculic acid to sterculicacid, and they postulated that the methylene group was derived frommethionine through S-adenosyl-methionine (see FIG. 1). They alsoindicated that sterculic acid was unlikely to be derived directly bymethylene addition across the 9,10-triple bond of stearolic acid,because no conversion of [1-¹⁴C] stearolic acid to sterculic acid wasobserved from the labeling experiments. Subsequently, there has beenessentially no further research into plant cyclopropane fatty acidsynthases.

It has been reported that, in Sterculia foetida seeds, total CPE-FAlevels were over 68% of total fatty acids, with small amounts ofdihydrosterculic acid also present (Badami and Patil (1981) Prog. LipidRes. 19: 119-153; Christie (1970) in Topics in Lipid Chemistry (GunstoneF D Ed.; Logos Press: London) Vol. 1, pp1-49; Sebedio and Gradgirard(1989) Prog. Lipid Res. 28:303). With oil content about 55% of its dryweight, Sterculia developing seeds appeared to be an ideal tissue tostudy the biosynthesis of CPE-FAs. Moreover, based upon severalassumptions, it was reasoned that Sterculia seed would be a good sourceof a plant cyclopropane fatty acid synthase (CPA-FAS). It was inferredthat the biosynthesis of sterculic acid occurs by a two step reaction,catalyzed by two separate enzymes, namely cyclopropane synthase andcyclopropane desaturase; this inference is based upon the in vivolabeling experiments (Yano et al (1972) Lipids 7: 35-45), and theknowledge of cyclopropane synthesis in bacteria. It was also inferredthat the cyclopropane synthase from Sterculia would share certain somedegree of similarity with bacterial cyclopropane synthase. Moreover,because Sterculia seeds have high levels of oil, and the oil has highlevels of high CPE-FAs, it was inferred that the transcript levels ofthe enzymes responsible for the synthesis of CPE-FAs should bereasonably high in developing seed tissue as well.

The first line of evidence that Sterculia seeds contained high levels ofthe enzymes required for CPA-FA and CPE-FA synthesis was based upon invivo labeling studies of developing Sterculia seed homogenates (seeExample 2). When samples of these homogenates were incubated withlabeled S-adenosyl methionine (SAM) and the lipid products saponified,labeled free fatty acid was the major constituent in the saponifiedproduct (greater than 90%). When analyzed after derivatization withethereal diazomethane and separation by C18 reversed-phase TLC, a singleradioactive spot co-eluted with the methyl dihycrosterculate standard.Thus, it was shown the a cell-free extract from developing Sterculiafoetida seeds could add a labeled methylene group fromS-adenosyl-methionine to oleate to produce dihydrosterculate. Thisreaction has not been reported previously in plant extracts. Additionalexperiments indicated that the enzyme synthesizing CFA-FA was either amembrane-associated or an integral membrane protein, and thatoleoyl-phosphatidylcholine was the substrate for the enzyme. Thus, theseresults indicated that the enzyme synthesizing CPA-FA was a CPA-FAS withsome characteristics similar to that of the E. coli CPA-FAS.

Based upon this evidence, a strategy for identifying a plant CPA-FAS wasdeveloped. This strategy begins with the observation of the presence ofCPA-FAs or CPE-FAs in a plant tissue. The next step is labeling studiesof tissue homogenates, to confirm that the ability to synthesize CPA-FAsand/or CPE-FAs is in fact present in the tissue. The next step isobtaining a large number of seed-specific ESTs by utilizing a cDNAlibrary prepared from the tissue (which for Sterculia is the developingseeds) which synthesize cyclopropane fatty acid, preferably to arelatively high level. For Sterculia, the fatty acid profiles ofdeveloping seeds were analyzed, to determine the developmental stagewhen CPE-FAs accumulated at the highest rate; seeds obtained at thisdevelopmental stage are then used to prepare a cDNA library. A smallfirst subset of the initial set of clones (about 10%) are sequenced,from which a smaller subset (about 7%) are obtained with an averagereading length of about 500 bp. This smaller subset is BLAST searched todiscover and select abundant sequences (which for Sterculia cDNA libraryrepresented about 30% of the clones), which are then subtracted out ofthe remaining clones. The subtracted clones are then sequenced, and asecond subset selected with an average reading length of 500 bp. Theseare also subjected to BLAST searches, resulting in a smaller set of ESTswhich show a certain degree of similarity with bacterial cyclopropanesynthase. These EST sequences 15, are then compiled to identify at leastone putative plant CPA-FAS.

Next, at least one complete cDNA clone encoding a putative plant CPA-FASis compiled from overlapping clones, and used to confirm the identity ofthe encoded sequence as a plant CPA-FAS. Confirmation is obtained byexpression of the clone in either an in vitro or in vivo system, suchthat either CPA-FAs are produced only upon expression of the clone, orincreased amounts of CPA-FAs are produced only upon expression of theclone. Preferably, the system is in vivo, and the clone transfected intoand expressed in a host organism. More preferably, the system in one inwhich CPA-FAs are not normally produced, such as when the host organismis a yeast strain. Even more preferably, the system possesses a suitablesubstrate, such as oleic acid, and is able to tolerate the presence ofunusual fatty acids, such as when the host organism is cultured tobaccocells.

This strategy was utilized for developing Sterculia seeds, as describedin the Examples, and resulted in the identification of 23 ESTs derivedfrom the same gene which were found to share some similarity withbacterial cyclopropane synthase; the distribution of these ESTs alongthe gene is shown in FIG. 3. The relative transcript abundance of thisgene is 0.36%, which is consistent with the 68% of CPE-FA content inSterculia seeds. A full length clone was assembled from the ESTscomprising SEQ ID NO:1 (as shown in FIG. 4). The predicted protein is864 amino acids long (SEQ ID NO:2, as shown in FIG. 5). Thus, thisprotein is about 470 amino acids longer than the E. coli CPA-FAS. TheSterculia CPA-FAS is 49% similar to and 32% identical to the E. colisequence over the region of overlap, which is the carboxy terminus. TheSterculia CPA-FAS thus has an additional approximately 470 amino acidsat the amino terminus.

Expression of the plant CPA-FAS in yeast and tobacco suspension cellsresulted in the synthesis of dihydrosterculic acid in both systems, andespecially in tobacco cells, where the amount of CPA-FA was high as 6.3%of total fatty acids. The radioactivity from both [1-¹⁴C] oleic acid andL-[methyl-¹⁴C] methionine were effectively incorporated intodihydrosterculic acids in the transgenic tobacco cells (see Example 3).These labeling results show that the biosynthesis of dihydrosterculicacid is through the addition a methylene group to the oleic acid acrossthe double bond. The methylene group is derived from methionine, mostlikely through S-adenosyl-methionine. In summary, these data clearlyconfirm that the identified Sterculia gene encodes a protein whichfunctions as a cyclopropane synthase.

Although it is not necessary to understand the mechanism to practice thepresent invention, and the invention is not intended to be limited toany particular mechanism, the following discussion of CPA-FAS and itsprotein structure and proposed and hypothetical functions providesfurther insight into the biology of the newly discovered protein. Sofar, one cyclopropane synthase from Escherichia coli (Wang et al (1992)Biochem 31 (45): 11020-11028) and three (cma1, cma2, and mma2) closelyrelated from mycobacterial (George et al. (1995) J Biol Chem 270 (45):27292-27298; and Yuan and Barry (1995) Proc Nat'l Acad Sci USA 92 (14):6630-6634) have been functionally proven to catalyze the transfer of amethylene group from S-adenosyl-L-methionine to the double bond ofcertain fatty acid. The Sterculia CPA-FAS reported here is the firstenzyme of this kind that has been isolated from plants. Another set ofgenes (mma1, mma3, and mma4) of mycobacteria is also highly homologousto E. coli CFA synthase. But these enzymes convert a double bond to oneof several structures (Yuan et al. (1997) J. Biol. Chem. 272:10041-10049), namely an alpha-methyl-branched trans-olefin—CH(CH3)CH:CH— (mmas-1) or an alpha-hydroxy-methyl (mmas-4)—CH(OH)CH(CH3)—. The alpha-hydroxymethyl structure can be converted toan alpha-methoxymethyl group —CH(OCH3)CH(CH3)— by addition of anothermethylene group, this time to the hydroxyl oxygene atom and not to anunsaturated carbon atom. Furthermore 10-methylstearate, ortuberculostearate, is a well known fatty acid of mycobacteria and isproduced by a 10-methylene stearate intermediate. The mechanisticsimilarities of these reactions are described by Grogan and Cronan(1997, Microbiol. Mol. Biol. Rev. 61: 429-441). They are readilyunderstood at the chemical level because the intermediate carbocationformed by addition of the methyl group from S-adenosyl-methionine canreadily rearrange, such that small changes in the active siteconfiguration can result in different structures of products.

A comparison of the amino acid sequence of Sterculia CPA-FAS with otherCPA-FASs and the related methoxy mycolic acid synthases is shown in FIG.10(A). All the bacterial amino acid sequences are about half the size ofSterculia cyclopropane synthase and share significant degree ofsimilarity with the carboxy terminus of Sterculia gene. The proposedS-adenosyl-methionine binding motif (amino acid residues 171-179, usingthe E. coli numbering, and amino residues 627-635 in the Sterculiaenzyme) and the catalytically important cysteine 354 (amino residue 822in the Sterculia enzyme) are absolutely conserved for all the proteins.FIG. 10(B) shows the phylogenetic relationship among these enzymes. TheSterculia and E. coli enzymes are more closely related to each otherthan to those enzymes from mycobacteria, which might reflect the factthat both enzymes act on monounsaturated fatty acids esterified tophospholipids (Ohlrogge et al. (1976) Biochim. Biophys. Acta 431:257-267). However, the fact that a highly related set of microbial genesencode a series of different methylene-added fatty acid synthasessuggests that the plant CPA-FAS gene could be modified to produce anequal diversity of products, thus enhancing its utility.

The amino terminal portion of the Sterculia CPA-FAS polypeptide (aminoacids 1-438) is unique to the Sterculia cyclopropane synthase, in thatno other known cyclopropane synthases possess this portion. It also doesnot share a significant similarity with any known proteins. However, theN-terminal half of Sterculia cyclopropane synthase shares significanthomology with Arabidopsis gene At3g23500, and to a lesser extent withAt3g23520. These putative genes encode products tentatively identifiedas tryptophan 2-monooxygenases. Tryptophan 2-monooxygenases belong to aflavin-containing group of oxidases. The tryptophan 2-monoxygenase(IaaM) gene product itself produces indole acetamide, which can beconverted to the auxin indole acetic acid by hydrolysis. Most offlavin-containing proteins have a highly conserved motif located theN-terminus of the protein and involved in binding of the ADP moiety ofFAD (Eggink et al, 1990; Eberhardt et al., 1996; Haigler et al, 1996).The proposed motif (G-X-G-X-X-G-X-X-X-A) is preceded by 3 or 4hydrophobic residues (Russel, M and Model, P (1988) J. Biol. Chem. 263:9015-9019.). The conserved FAD binding motif is present in the first 15amino acid of Sterculia cyclopropane synthase(MGVAVIGGGIQGLVSAYVLAKAGVNVVVYE).

Since the mechanism of the cyclopropane ring formation is believed toproceed via a carbocation mechanism, with the proposed intermediate(—CHCH₃—CH⁺—) formed by addition of the methyl group fromS-adenosylmethionine, it is unlikely that a redox system such as anFAD-containing protein is involved in the catalytic reaction ofmethylene addition. Therefore, it is unlikely that the amino terminalportion of the Sterculia CPA-FAS is involved in cyclopropane ringformation. It appears that the CPA-FA synthase polypeptide contains afused redox protein at its amino terminus (approximately amino acids1438), which is probably an oxidase. It is contemplated that the redoxprotein domain functions either in the desaturation of dihydrosterculicacid to sterculic acid, or more likely in the ∀-oxidation thataccompanies the formation of sterculic acid.

It is believed that dihydrosterculic acid is converted to sterculic acidby desaturation (FIG. 1). In labeling studies with developing Sterculiaseed tissue, added [¹⁴C]stearolate, the 9,10-acetylenic analog ofoleate, was not converted to [¹⁴C]sterculate. On the other hand,dihydrosterculate was clearly desaturated to sterculate, but theconversion rates were slow. Nonetheless, taken together, these datasupport the proposed pathway for the synthesis of sterculic acid bydesaturation of dihydrosterculic acid. However, an unusual featureassociated with sterculic acid biosynthesis in both seeds and otherplant tissues is substantial ∀-oxidation. Often malvalic acid is themajor CPE-FA. Although other non-carbocyclic fatty acids show a smallamount of ∀-oxidation products, the CPE-FA are often chain-shortened toa much greater proportion. Such extensive ∀-oxidation seems unique toCPE-FA biosynthesis. However, in the tobacco cell lines transformed withthe coding sequence for Sterculia CPA-FAS, no 17- or 18-carbon CPA-FAswere found in any independent transgenic tobacco cell line screened. Thelack of 17 carbon CPA-FAs is thought to be due to the lack of 16:1substrate. The lack of 18 carbon CPA-FAs is an indication that theSterculia CPA-FAS did not induce ∀-oxidation, despite the presence ofthe apparent redox fusion protein, which is contemplated to be involvedin ∀-oxidation. However, it is contemplated that the substrate of∀-oxidation is the unsaturated CPE-FA, rather than the saturated CPA-FA.Support is provided by the observation that in Litchi, the only seedknown where CPA-FAs accumulate without CPE-FA, there are no ∀-oxidationproducts of dihydrosterculic acid (though traces of 17- and 5-carbon∃-oxidation products are seen; Gaydou et al. (1993) J. Agric. Food Chem.41: 886-890).

Thus, it is contemplated that the Sterculia CPA-FAS comprises a naturalfusion product of two catalytic activities; one catalyzes thecyclopropane fatty acid synthesis (the carboxy terminal, at about aminoacids 439-864), and the other catalyzes an ∀-oxidation shortening of afatty acid with a cyclic carbon functional group, most likely anunsaturated cyclic carbon ring or a cyclopropene ring (the aminoterminal, at about amino acids 1438). Examples of fusion proteins, andin particular of fusion of proteins involved in lipid synthesis, areknown (for example, a single polypeptide with two enzymatic activitieswas reported to occur naturally in coral, where a fusion proteincontains both lipoxygenase and allene oxide synthase (Koljak et al.(1997) Science 277: 1994-1996; Boutand and Brash (1999) J. Biol. Chem.274: 33764-33770)

The Sterculia CPA-FAS is the first confirmed identification of a plantCPA-FAS, despite one recent report of the putative identification ofplant nucleic acid sequences which purportedly encode cyclopropanesynthetase (or CFA synthase) (WO 99/43827). The application describesnucleic acid fragments reportedly encoding at least a portion of severalcyclopropane synthetases; these fragments were isolated and identifiedby comparison of random plant cDNA sequences to public databasescontaining nucleotide and protein sequences using BLAST algorithms. Theencoded proteins were discovered based upon their similarity tocyclopropane synthetase from Mycobacterium tuberculosis or from E. coli.The results are a set of five nucleotide sequences, one of which is acontig from corn (assembled from three clones), the next three of whichare clones from Phaseolus, rice and soybean, and the last of which is acontig from wheat (assembled from two clones); only the first twosequences appear to encode a “complete” protein of about 386 aminoacids. The first “complete” protein is described as containing a signalsequence of amino acids 1-28 (though how this signal sequence wasidentified was not described) and a mature protein of amino acids29-386. The E. coli CFA synthase amino acid sequence is also describedas 19.95% and 19.2% similar to the two “complete” proteins,respectively. The application does not provide any evidence that thesequences in fact encode a plant CPA-FAS, other than the homology theamino acid sequences (as predicted from the nucleic acid sequences)exhibit to the bacterial enzymes.

However, it appears that WO 99/43827 describes nucleic acid sequenceswhich do not actually encode plant CFA synthases. This is based uponseveral lines of reasoning. First, the predicted amino acid sequence ofthe Sterculia CFA synthase is 864 amino acids long, or more than twiceas long as the “complete” proteins described in WO 99/43827. The aminoacid sequence of the Sterculia enzyme appears to have a higher degree ofhomology to the E. coli sequence, at the region of overlap, than do the“complete” sequences described WO 99/43827. The present inventors haveprovided evidence in Example 3 that transformation of the Sterculianucleic acid sequence into yeast and plant cells results in theproduction of CPA-FAs in a tissue which does not normally have suchfatty acids; this is in contrast to WO 99/43827, which does not provideany evidence at all that expression of such sequences can result in theappearance of CPA-FAs. The source from which the Sterculia nucleic acidsequence was isolated was predicted to be an abundant source of theenzyme, based upon the presence of CPE-FAs of up to 60% in sterculiaseed oil, and based upon the assumption that CPE-FAs are derived fromCPA-FAs. This is in contrast to the sources from which the cDNAlibraries were prepared in WO 99/43827, which were corn, Phaseolus,rice, soybean, and wheat, and which are not known to contain CPA-FAs.Finally, there are many S-adenosyl methionine dependent methyltransferases with similar DNA and protein sequences (as described, forexample, in Wang et al. (1992) Biochemistry 31: 11020-11028). Thereforesequence similarity alone is not sufficient to demonstrate proteinfunction and identity.

However, the Sterculia CPA-FAS amino acid sequence can be used todiscover other plant CPA-FASs, as is described further below; in oneembodiment, coding sequences for cotton CPA-FAS are discovered by themethods described below, and as is described in further detail inExample 5.

A. Plant Cyclopropane Fatty Acid Synthase Genes

The present invention provides compositions comprising isolated nucleicacid sequences encoding plant CPA-FAS. In some embodiments, thesequences encode a Malvaceae CPA-FAS; in other embodiments, thesequences encode a Sterculia CPA-FAS; in yet other embodiments, thesequences encode a cotton CPA-FAS. In some embodiments, the sequencescomprise the sequence shown in FIG. 4 (SEQ ID NO: 1); in otherembodiments, the sequences encode the amino acid sequence shown in FIG.5 (SEQ ID NO:2). In yet other embodiments, the sequences comprise atleast one of the sequences shown in FIGS. 14 and 15 (SEQ ID NOs:3, 4, 5,and 6); in other embodiments, the sequences encode at least one of theamino acid sequences shown in FIG. 16 (SEQ ID NOs:7, 8, 9, and 10). Inpreferred embodiments, the CPA-FAS encoded by the nucleic acid sequencesof the invention are functional, or possess CPA-FAS activity.

In yet other embodiments, the present invention provides compositionscomprising isolated nucleic acid sequences which encode a portion of aplant CPA-FAS which retains some functional characteristic of a CPA-FAS.Examples of functional characteristics include the ability to act as animmunogen to produce an antibody which recognizes a CPA-FAS (see, forexample, Example 4); in particular embodiments, the nucleic acidsequences encode the amino acid sequence shown in FIG. 13 (SEQ ID NO:11) Other examples include nucleic acid sequences which encode eitherthe amino terminal or the carboxy terminal of a plant CPA-FAS, which arehypothesized to possess separate and distinct enzymatic capabilities; inthese embodiments, the nucleic acid sequences encode either the aminoterminal of a plant CPA-FAS (which in Sterculia is about amino acids1-438) or the carboxy terminal of a plant CPA-FAS (which in Sterculia isabout amino acids 439-864); in particular embodiments, these proteinfragments retain the enzymatic activity associated with it in the nativeor complete CPA-FAS protein. In other particular embodiments, thenucleic acid fragments encode SEQ ID NOs:7, 8, 9, or 10.

In yet other embodiments, the present invention provides compositionscomprising isolated nucleic acid sequences encoding a plant CPA-FAS,where the encoded CPA-FAS comprises at least one fragment of SEQ IDNOs:2, 7, 8, 9, or 10, and wherein the encoded CPA-FAS is cross-reactivewith an antibody against a Sterculia CPA-FAS and is about the same sizeas a Sterculia CPA-FAS (about 864 amino acids long). An exemplaryantibody to CPA-FAS is described in Example 4. The range of sizes of aSterculia CPA-FAS is from about 800-900 amino acids long. A fragment ofan amino acid sequence comprises at least 10, more preferably 20, evenmore preferably 30, and most preferably 40 or more amino acid residuespresent anywhere within the amino acid sequence. Alternatively, afragment comprises at least 20, more preferably about 30, even morepreferably about 40, and most preferably about 50 or more amino acidresidues present anywhere within the amino acid sequence, wherein thefragment further comprises at least one amino acid deletion or additionor substitution, where the number of total amino acid additions,deletions, or substitutions in any one fragment comprise up to about 10%of the total number of amino acids in the fragment. If more than oneamino acid deletion, addition, or substitution exist, they may becontiguous or non-contiguous. Preferably, amino acid substitutions areconservative.

B. Plant Cyclopropane Fatty Acid Synthase Polypeptides

The present invention provides compositions comprising purified plantCPA-FAS polypeptides as well as compositions comprising variants,including homologs, mutants or fragments, or fusion proteins thereof. Insome embodiments, the polypeptide comprises a Malvaceae CPA-FAS; inother embodiments, the polypeptide comprises a Sterculia CPA-FAS; in yetother embodiments, the polypeptide comprises a cotton CPA-FAS. In oneembodiment, the polypeptide is encoded by the sequence shown in FIG. 4(SEQ ID NO: 1); in other embodiments, the polypeptide comprises theamino acid sequence shown in FIG. 5 (SEQ ID NO:2). In other embodiments,the polypeptide is encoded by a sequence comprising at least one of thesequences shown in FIGS. 14 and 15 (SEQ ID NOs:3, 4, and 6); in otherembodiments, the polypeptide comprises at least one of the amino acidsequences shown in FIG. 16 (SEQ ID NOs:7 and 8).

The polypeptide catalyzes the addition of a methylene group across theunsaturated center of an unsaturated fatty acid, and includes theaddition of a methylene group across a double bond of an acyl group.Thus, a plant CPA-FAS of the present invention is a polypeptide with thecapacity to synthesize a fatty acid containing a cyclopropane ring.

Thus, plant CPA-FAS catalyzes the following reaction:unsaturated fatty acyl-X+CH₂→cyclopropane fatty acyl-Xwhere X is preferably a glycerolipid, and most likely a phospholipid,such as phosphatidylcholine. The enzyme in situ most likely acts on afatty acid such as oleic or palmitoleic acid esterified to a lipid, anduses S-adenosyl methionine (SAM) as a methyl donor. Moreover, the enzymemay utilize different substrates under different conditions to differingdegrees of activity, and may produce other substrates as well. Thus,other substrates may accept a methylene group, and the resulting fattyacyl group may have an unsaturated carbocyclic ring.

In some embodiments of the present invention, the polypeptide is apurified product, obtained from expression of a native gene in a cell,while in other embodiments it may be a product of chemical syntheticprocedures, and in still other embodiments it may be produced byrecombinant techniques using a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). In some embodiments, depending upon the host employed in arecombinant production procedure, the polypeptide of the presentinvention may be glycosylated or may be non-glycosylated. In otherembodiments, the polypeptides of the invention may also include aninitial methionine amino acid residue.

Assay of Plant Cyclopropane Fatty Acid Synthase

The activity of plant CPA-FAS may be assayed in a number of ways. In oneaspect, the activity is determined by expressing a nucleic acid sequenceencoding the synthase in a transgenic organism and then analyzing thecomposition of the total fatty acids. Thus, the activity is measured asthe presence of or increase in the amount of endogenous cyclopropanefatty acid in a transgenic organism which comprises an exogenous nucleicacid sequence comprising SEQ ID NO: 1 or encoding a polypeptidecomprising the amino acid sequence shown in SEQ ID NO:2; such transgenicorganisms are obtained as described elsewhere The amount of cyclopropanefatty acid in a transgenic organism is compared to that present in anon-transgenic organism. The fatty acids are typically analyzed fromlipids extracted from samples of a transgenic organism; the samples arehomogenized in methanol/chloroform (2:1, v/v) and the lipids extractedas described by Bligh and Dyer (1959).

In another aspect, the enzyme activity is determined in tissue samplesobtained from a organism which may or may not be transgenic. Forexample, in plants, tissue samples include but are not limited to leafsamples (such as discs), stem and root samples, and developing andmature seed embryonic or endosperm tissue. Typically, tissue samples areincubated with either precursors of fatty acid synthesis, such as¹⁴C-acetate, or with fatty acid substrates, such as ammonium salts of¹⁴C-fatty acids, which can be taken up and incorporated into tissuelipids, or with labeled substrates, such as ¹⁴C-methionine. Additionalco-factors for lipid synthesis, as required, are present during theincubation; such co-factors include but are not limited to ATP, CoA,Mgl₂, and SAM. Alternatively, tissue samples are incubated with labeledSAM: Incubations generally proceed at room temperature in a bufferedsolution, such as 0.1M potassium phosphate at pH 7.2, for a suitableperiod of time. The samples are then washed in buffer, and the tissuesamples homogenized in methanol/chloroform (2:1, v/v) and the lipidsextracted as described by Bligh and Dyer (1959).

In another aspect, the enzyme activity is determined in a sub-cellularfraction obtained from an organism which may or may not be transgenic(transgenic organisms are described elsewhere), where the tissue isdisrupted to result in cell-free fractions. For example, in plants,subcellular fractions may be obtained from any of the types of tissuesdescribed above, and include whole cell and microsomal membranes,plastids, and plastidial membrane fractions. Preparation of suchfractions are well-known in the art. The subcellular fraction is thenincubated with fatty acids, such as ammonium salts of ¹⁴C-fatty acids,which can be taken up and incorporated into tissue lipids.Alternatively, the subcellular fraction is incubated with labeled SAM.Additional co-factors for lipid synthesis, as required, are presentduring the incubation; such co-factors include but are not limited toATP, CoA, MgCl₂, lyso-phospholipids, such as lysoPC, and SAM. Otherreagents which may enhance lipid synthesis may also be added; suchreagents include phospholipid liposomes (for example, containingphosphatidylcholine) and lipid transfer proteins. The samples areincubated and the lipids extracted as described above.

In another aspect, the enzyme activity is determined from an in-vitronucleic acid expression system, in which a nucleic acid sequencecomprising SEQ ID NO: 1 or encoding a polypeptide comprising the aminoacid sequence shown in SEQ ID NO:2 is added and the encoded enzymeexpressed. Such expression systems are well-known in the art, forexample reticulocyte lysate or wheat germ. Because the enzyme is likelyto be an unstable protein which is stabilized by the presence ofglycerolipids, micellar or membrane structures are included in themixture into which the enzyme may be incorporated during or afterprotein synthesis. Moreover, because the enzyme in situ is likely to acton a fatty acid esterified to a lipid, it is preferable that suchmicellar structures are obtained from sources which contain relatedlipid synthetic capabilities, such as from microsomes from plant tissueswhere the plant does not contain an endogenous cyclopropane fatty acidsynthase but which does possess the ability to incorporate a labeledfatty acid substrate into a glycerolipid. Direct and quantitativemeasurements require the incorporation of labeled lipids into themicellar or membrane structures and the assurance that the incorporationof a fatty acid substrate is not limiting. The newly-expressed enzyme isthen analyzed as described above for subcellular fractions.

The extracted lipid products of the plant CPA-FAS are analyzed bymethods well-known in the art. For example, fatty acid methyl esters areprepared from an aliquot of the extracted lipid fraction by evaporatingthe solvent from the aliquot under N₂, and resuspending the lipids inequal volumes of 1% sodium methoxide in methanol (w/w) and heptane.Cyclopropane and cyclopropene groups in fatty acids are disrupted byacidic conditions, and lipid samples containing such acids are besttransesterified with basic reagents; the free fatty acids can bemethylated safely with diazomethane (Christie (1982) in Lipid Analysis,2nd Ed., p 55). The fatty acid methyl esters are then extracted intohexane and separated, and for radioactive samples the radioactivity ineach separated fraction determined, by TLC, GC, or GC/MS (see, forexample, described in Example 1).

Purification of Plant Cyclopropane Fatty Acid Synthase

In some embodiments of the present invention, a plant CPA-FASpolypeptide purified from organisms is provided; such organisms may betransgenic organism, comprising a heterologous plant CPA-FAS gene. Thepresent invention provides a purified plant CPA-FAS polypeptide as wellas a variant, homolog, mutant or fusion protein thereof, as describedelsewhere.

The present invention also provides methods for recovering and purifyingplant CPA-FAS from an organism; such organisms include single andmulti-cellular organisms. Typically, the cells are first disrupted andfractionated before subsequent enzyme purification; disruption andfractionation methods are well-known. Purification methods are alsowell-known, and include, but are not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography.

It is anticipated that plant CPA-FAS is unstable, by analogy with E.coli cyclopropane fatty acid synthase (CFA synthase) (Grogan, D W andCronan, J W Jr. (1997) Microbiol Molec Biol Reviews 61(4): 429441). TheE. coli enzyme is extremely unstable, such that when crude extracts ofE. coli were freed of endogenous lipid by ultracentrifugation, less than1% of the initial CFA synthase activity remained after a 30-minincubation at 30″C, and that this instability has severely hamperedpurification and detailed studies of the enzyme. It was noted that theenzyme associates reversibly with membrane fragments and withphospholipid vesicles, and that this association is the only generallyeffective means of stabilizing CFA synthase identified at the time thereference was published. Therefore, it is contemplated that in oneembodiment, a plant CPA-FAS of the present invention is purified asdescribed for bacterial CFA synthase (Grogan, D W and Cronan, J W Jr.(1997) Microbiol Molec Biol Reviews 61(4): 429441). This scheme involvesdisruption of cells (as for example by passage through a French press orby homogenization), centrifugation at high speeds to remove cellulardebris (as for example at 150,000 g for about 2 hours), precipitation ofprotein from the resulting supernatant, as for example by addingammonium sulfate to about 40% saturation, collecting the precipitatedprotein by centrifugation (as for example at 10,000 g for about 15 min),removal of residual ammonium sulfate from the resuspended protein pellet(as for example by gel filtration), and liposome flotation of the enzymeand subsequent purification of the lipid layer by sucrose gradientcentrifugation.

The present invention further provides nucleic acid sequences having acoding sequence of the present invention (for example, SEQ ID NO: 1)fused in frame to a marker sequence that allows for expression alone orboth expression and purification of the polypeptide of the presentinvention. A non-limiting example of a marker sequence is ahexahistidine tag that may be supplied by a vector, for example, apQE-30 vector which adds a hexahistidine tag to the N terminal of aplant CPA-FAS and which results in expression of the polypeptide in thecase of a bacterial host, and more preferably by vector PT-23B, whichadds a hexahistidine tag to the C terminal of a plant CPA-FAS and whichresults in improved ease of purification of the polypeptide fused to themarker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host is used.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al. (1984) Cell, 37:767).

Chemical Synthesis of Plant Cyclopropane Fatty Acid Synthase

In an alternate embodiment of the invention, the coding sequence of aplant CPA-FAS is synthesized, whole or in part, using chemical methodswell known in the art (See for example, Caruthers et al. (1980) Nucl.Acids Res. Symp. Ser., 7:215-233; Crea and Horn (1980) Nucl. Acids Res.,9:2331; Matteucci and Caruthers (1980) Tetrahedron Lett., 21:719; andChow and Kempe (1981) Nucl. Acids Res., 9:2807-2817). In otherembodiments of the present invention, the protein itself is producedusing chemical methods to synthesize either an entire plant CPA-FASamino acid sequence or a portion thereof. For example, peptides aresynthesized by solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography (See forexample, Creighton (1983) Proteins Structures And Molecular Principles,W H Freeman and Co, New York N.Y.). In other embodiments of the presentinvention, the composition of the synthetic peptides is confirmed byamino acid analysis or sequencing (See for example, Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al. (1995) Science, 269:202-204) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, an amino acid sequence of a plantCPA-FAS, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with other sequences to produce avariant polypeptide.

Generation of Plant Cyclopropane Fatty Acid Synthase Antibodies

In some embodiments of the present invention, antibodies are generatedto allow for the detection and characterization of a plant CPA-FASprotein. The antibodies may be prepared using various immunogens. In oneembodiment, the immunogen is a Sterculia CPA-FAS peptide (for example,an amino acid sequence as depicted in SEQ ID NO:2, or fragments thereof)to generate antibodies that recognize Sterculia CPA-FAS. Such antibodiesinclude, but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and Fab expression libraries.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against a plant CPA-PAS. For theproduction of antibody, various host animals can be immunized byinjection with the peptide corresponding to a plant CPA-FAS epitopeincluding but not limited to rabbits, mice, rats, sheep, goats, etc. Ina preferred embodiment, the peptide is conjugated to an immunogeniccarrier (for example, diphtheria toxoid, bovine serum albumin (BSA), orkeyhole limpet hemocyanin (KLH)). Various adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels (for example, aluminum hydroxide), surface active substances (forexample, lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanins, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (Bacille Calmette-Guerin) andCorynebacterium parvum).

For preparation of monoclonal antibodies directed toward a plantCPA-FAS, it is contemplated that any technique that provides for theproduction of antibody molecules by continuous cell lines in culturefinds use with the present invention (See for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). These include but are not limited to thehybridoma technique originally developed by Köhler and Milstein (Köhlerand Milstein (1975) Nature, 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (See for example, Kozboret al. (1983) Immunol. Tod., 4:72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al. (1985) in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) find usein producing a plant CPA-FAS-specific single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab expression libraries (Huse et al. (1989)Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for a plantCPA-FAS.

It is contemplated that any technique suitable for producing antibodyfragments finds use in generating antibody fragments that contain theidiotype (antigen binding region) of the antibody molecule. For example,such fragments include but are not limited to: F(ab′)2 fragment that canbe produced by pepsin digestion of the antibody molecule; Fab′ fragmentsthat can be generated by reducing the disulfide bridges of the F(ab′)2fragment, and Fab fragments that can be generated by treating theantibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody is accomplished by techniques known in the art (forexample, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays (forexample, using colloidal gold, enzyme or radioisotope labels, forexample), Western blots, precipitation reactions, agglutination assays(for example, gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.

In some embodiments of the present invention, the foregoing antibodiesare used in methods known in the art relating to the expression of plantCPA-FAS (for example, for Western blotting), measuring levels thereof inappropriate biological samples, etc. The antibodies can be used todetect plant CPA-FAS in a biological sample from a plant. The biologicalsample can be an extract of a tissue, or a sample fixed for microscopicexamination.

The biological samples are then be tested directly for the presence ofplant CPA-FAS using an appropriate strategy (for example, ELISA orradioimmunoassay) and format (for example, microwells, dipstick (forexample, as described in International Patent Publication WO 93/03367),etc. Alternatively, proteins in the sample can be size separated (forexample, by polyacrylamide gel electrophoresis (PAGE), in the presenceor not of sodium dodecyl sulfate (SDS), and the presence of plantCPA-FAS detected by immunoblotting (Western blotting). Immunoblottingtechniques are generally more effective with antibodies generatedagainst a peptide corresponding to an epitope of a protein, and hence,are particularly suited to the present invention.

II. Methods of Identifying Plant CPA-FAS Genes and Related Plant Genes

Some embodiments of the present invention contemplate methods to isolatenucleic acid sequences encoding plant CPA-FAS, based upon the hypothesisthat the presence of CPA-FAs and/or CPE-FAs in plant tissue, preferablyseed tissue, is indicative of the presence of CPA-FAS. The methodsinvolve first preparation of a cDNA library from tissue in which CPA-FAsor CPE-FAs are produced to relatively high levels. The methods involvenext subtracting highly abundant sequences from the library, sequencingthe remaining library clones, and comparing the encoded amino acidsequences to the amino acid sequence of either E. coli CPA-FAS orSterculia CPA-FAS to select putative CPA-FAS candidate ESTs. The methodsinvolve next assembling a clone encoding a complete putative plantCPA-FAS, and characterizing the expression products of such sequences sodiscovered.

Alternatively, the methods involve first an examination of a plantexpressed sequence tag (EST) database, in order to discover novelpotential CPA-FAS encoding sequences. Preferably, the plant source ofthe EST database comprises CPA-FAs and/or CPE-FAs in its plant tissue,such as its seed tissue. In some embodiments, examination of a plant ESTdatabase involves blasting the database with the amino acid sequence ofthe Sterculia CPA-FAS (for example, SEQ ID NO:2), in order to discoverESTs encoding amino acid sequences with homology to the SterculiaCPA-FAS protein. In some further embodiments, the methods involve nextassembling a clone encoding a complete putative plant CPA-FAS, andcharacterizing the expression products of such sequences so discovered.In other further embodiments, these methods next involve sequencinglikely candidate sequences, and characterizing the expression productsof such sequences so discovered.

Employing these methods resulted in the discovery of a SterculiaCPA-FAS, as described in illustrative Examples. The isolated novelcoding sequence was demonstrated to encode plant cyclopropane fatty acidsynthase, as described in the illustrative Examples. It is contemplatedthat these methods can also be used to discover other CPA-FASs fromplants which are known to possess CPA-FAs and CPE-FAs. Exemplary plantsinclude those from the families Malvaceae, Sterculiaceae, Bombaceae,Tilaceae, Mimosaceae and Sapindaceae. In particular, it is contemplatedthat a CPA-FAS from cotton is identified and isolated by these methods.Thus, employing these methods resulted in the discovery of cottonCPA-FAS coding sequences, as described in illustrative Examples. Cottontissues were demonstrated to contain cyclopropane and cyclopropene fattyacids (CPA-FAs and CPE-FAs), where certain tissues (such as root andstem) contained relatively high levels of these fatty acids (up to about30% and about 35%, respectively). Moreover, cotton tissues weredemonstrated to contain a protein which cross-reacts with antibodyprepared to the Sterculia CPA-FAS, where the protein is about the samesize as the Sterculia CPA-FAS, and where the protein has a tissuedistribution which generally corresponds to the amounts of CPA-FAs andCPE-FAs present in the tissues.

The nucleotide sequence encoding the Sterculia CPA-FAS, and the deducedamino acid sequence of the Sterculia CPA-FAS, are shown in FIG. 4 (SEQID NOs 1 and 2, respectively). The Sterculia CPA-FAS coding sequence canbe used to locate and isolate the Sterculia CPA-FAS genes, by methodswell known in the art; thus, plant CPA-FAS coding sequences, discoveredby the methods of the present invention, can also be used to locate andisolate other plant genes by these same methods. To isolate the gene, a³²P-radiolabeled CPA-FAS coding sequence (or cDNA) is used to screen, byDNA-DNA hybridization, a genomic or cDNA library constructed fromSterculia genomic DNA. Single isolated clones that test positive forhybridization are proposed to contain part or all of the CPA-FAS gene,and are sequenced. The sequence of these positive cloned Sterculiagenomic DNA is used to confirm the identity of the gene as a plantCPA-FAS. If a particular clone encodes only part of the gene, additionalclones that test positive for hybridization to the CPA-FAS codingsequence (or cDNA) are isolated and sequenced. Comparison of thefull-length sequence of the CPA-FAS gene to the cDNA are used todetermine the location of introns, if they are present.

The Sterculia CPA-FAS can also be used to identify and isolate relatedplant genes. As an example, it is believed that CPE-FA is synthesizedvia desaturation of CPA-FA. Although tobacco cell lines transformed withSterculia CPA-FAS produce CPA-FA (dihydrosterculic acid), they do notappear to produce CPE-FA (sterculic acid). Therefore, it appears thatthe identified Sterculia CPA-FAS does not desaturate CPA-FA to CPE-FA,and that another Sterculia polypeptide is responsible for desaturatingCPA-FA. A nucleic acid sequence for this activity is identified andisolated according to the methods described above for identifyingCPA-FAS coding sequences, except that desaturase amino acid sequencesare used as the basis for homology comparisons. It is contemplated thatthe desaturase is homologous to a FAD2 or a P450 enzyme. Candidatesequences are then co-transformed into tobacco cell lines alreadytransformed with Sterculia CPA-FAS, as for example is described in theExamples, and the fatty acid products analyzed, as for example isdescribed in the Examples. The presence of CPE-FA in the transgenic celllines confirms that the candidate sequence is a CPA-FA desaturase.

III. Additional Plant Cyclopropane Fatty Acid Synthase Genes

The present invention provides isolated nucleic acid sequences encodinga plant CPA-FAS. For example, some embodiments of the present inventionprovide isolated polynucleotide sequences that are capable ofhybridizing to SEQ ID NOs: 1, 3, and/or 6 under conditions of low tohigh stringency as long as the polynucleotide sequence capable ofhybridizing encodes a protein that retains a desired biological activityof a plant CPA-FAS. In preferred embodiments, hybridization conditionsare based on the melting temperature (T_(m)) of the nucleic acid bindingcomplex and confer a defined “stringency” as explained above (See forexample, Wahl et al. (1987) Meth. Enzymol., 152:399-407, incorporatedherein by reference).

In other embodiments, an isolated nucleic acid sequence encoding a plantCPA-FAS which is homologous to the Sterculia CPA-FAS is provided; insome embodiments, the sequence is obtained from a plant from the familyMalvaceae, Sterculiaceae, Bombaceae, Tilaceae, Mimosaceae orSapindaceae. In particular embodiments, such sequences are obtained fromcotton; these sequences comprise at least one of SEQ ID NOs:3, 4 and 6.

In other embodiments of the present invention, alleles of a plantCPA-FAS are provided. In preferred embodiments, alleles result from amutation, (in other words, a change in the nucleic acid sequence) andgenerally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes that give rise to allelesare generally ascribed to deletions, additions or substitutions ofnucleic acids. Each of these types of changes may occur alone, or incombination with the others, and at the rate of one or more times in agiven sequence.

In other embodiments of the present invention, the polynucleotidesequence encoding a plant CPA-FAS is extended utilizing the nucleotidesequences for example, SEQ ID NO: 1) in various methods known in the artto detect upstream sequences such as promoters and regulatory elements.For example, it is contemplated that polymerase chain reaction (PCR)finds use in the present invention. This is a direct method that usesuniversal primers to retrieve unknown sequence adjacent to a known locus(Gobinda et al. (1993) PCR Methods Applic., 2:318-322). First, genomicDNA is amplified in the presence of primer to a linker sequence and aprimer specific to the known region. The amplified sequences are thensubjected to a second round of PCR with the same linker primer andanother specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase andsequenced using reverse transcriptase.

In another embodiment, inverse PCR is used to amplify or extendsequences using divergent primers based on a known region (Triglia et al(1988) Nucleic Acids Res., 16:8186). The primers may be designed usingOligo 4.0 (National Biosciences Inc, Plymouth Minn.), or anotherappropriate program, to be, for example, 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68-72 EC. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template. In yet another embodiment of the present invention,capture PCR (Lagerstrom et al. (1991) PCR Methods Applic., 1:111-119) isused. This is a method for PCR amplification of DNA fragments adjacentto a known sequence in human and yeast artificial chromosome (YAC) DNA.Capture PCR also requires multiple restriction enzyme digestions andligations to place an engineered double-stranded sequence into anunknown portion of the DNA molecule before PCR. In still otherembodiments, walking PCR is utilized. Walking PCR is a method fortargeted gene walking that permits retrieval of unknown sequence (Parkeret al. (1991) Nucleic Acids Res., 19:3055-60). The PROMOTERFINDER kit(Clontech) uses PCR, nested primers and special libraries to “walk in”genomic DNA. This process avoids the need to screen libraries and isuseful in finding intron/exon junctions. In yet other embodiments of thepresent invention, add TAIL PCR is used as a preferred method forobtaining flanking genomic regions, including regulatory regions (Luiand Whittier, (1995); Lui et al. (1995)).

Preferred libraries for screening for full length cDNAs includelibraries that have been size-selected to include larger cDNAs. Also,random primed libraries are preferred, in that they contain moresequences that contain the 5′ and upstream gene regions. A randomlyprimed library may be particularly useful in cases where an oligo d(T)library does not yield full-length cDNA. Genomic Libraries are usefulfor obtaining introns and extending 5′ sequence.

IV. Variant Plant Cyclopropane Fatty Acid Synthases

In some embodiments, the present invention provides isolated variants ofthe disclosed nucleic acid sequence encoding plant CPA-FAS, and thepolypeptides encoded thereby; these variants include mutants, fragments,fusion proteins or functional equivalents of plant CPA-FAS. Thus,nucleotide sequences of the present invention are engineered in order toalter a plant CPA-FAS coding sequence for a variety of reasons,including but not limited to alterations that modify the cloning,processing and/or expression of the gene product (such alterationsinclude inserting new restriction sites, altering glycosylationpatterns, and changing codon preference) as well as varying theenzymatic activity (such changes include but are not limited todiffering substrate affinities, differing substrate preferences andutilization, differing inhibitor affinities or effectiveness, differingreaction kinetics, varying subcellular localization, and varying proteinprocessing and/or stability). For example, mutations are introducedwhich alter the substrate specificity, such that the preferred substrateis changed.

In other embodiments, the present invention provides isolated nucleicacid sequences encoding a plant CPA-FAS, where the encoded synthasecompetes for binding to an unsaturated fatty acid substrate with aprotein comprising the amino acid sequence of SEQ ID NO:2.

Mutants of a Plant Cyclopropane Synthase

Some embodiments of the present invention provide mutant forms of aplant CPA-FAS (in other words, muteins). In preferred embodiments,variants result from mutation, (in other words, a change in the nucleicacid sequence) and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one, or many mutant forms. Common mutational changes that giverise to variants are generally ascribed to deletions, additions orsubstitutions of nucleic acids. Each of these types of changes may occuralone, or in combination with the others, and at the rate of one or moretimes in a given sequence.

It is contemplated that is possible to modify the structure of a peptidehaving an activity (for example, a plant CPA-FAS activity) for suchpurposes as increasing synthetic activity or altering the affinity ofthe plant CPA-FAS for a particular fatty acid substrate. Such modifiedpeptides are considered functional equivalents of peptides having anactivity of a plant CPA-FAS as defined herein. A modified peptide can beproduced in which the nucleotide sequence encoding the polypeptide hasbeen altered, such as by substitution, deletion, or addition. In somepreferred embodiments of the present invention, the alteration increasessynthetic activity or alters the affinity of the plant CPA-FAS for aparticular fatty acid substrate. In particularly preferred embodiments,these modifications do not significantly reduce the synthetic activityof the modified enzyme. In other words, construct “X” can be evaluatedin order to determine whether it is a member of the genus of modified orvariant plant CPA-FAS of the present invention as defined functionally,rather than structurally. In preferred embodiments, the activity ofvariant plant CPA-FAS is evaluated by the methods described in Example3. Accordingly, in some embodiments the present invention providesnucleic acids encoding a plant CPA-FAS that complement the coding regionof SEQ ID NO: 1. In other embodiments, the present invention providesnucleic acids encoding a plant CPA-FAS that compete for the binding offatty acid substrates with the protein encoded by SEQ ID NO: 1.

In one embodiment, site-specific mutagenesis is performed to modify thecatalytic activity of a plant CPA-FAS from a methylene addition acrossthe double bond to a methylene addition at the 10-position, as is knownto occur for the synthesis of tubercuolic acid. The modified enzyme willproduce fatty acids that have pendant vinyl groups, which is a valuableplatform for industrial derivatization. Moreover, hydrogenation of thefatty acid products will give quantitative conversion to methyl-branchedsaturates.

As described above, mutant forms of a plant CPA-FAS are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (in other words, conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Accordingly, some embodiments of the present invention provide variantsof a plant CPA-FAS disclosed herein containing conservativereplacements. Conservative replacements are those that take place withina family of amino acids that are related in their side chains.Genetically encoded amino acids can be divided into four families: (1)acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine);(3) nonpolar (alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); and (4) uncharged polar(glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (for example, Stryer ed.(1981) Biochemistry pg. 17-21, 2nd ed, WH Freeman and Co.). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (for example,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (for example, LASERGENE software, DNASTAR Inc.,Madison, Wis.).

Mutants of a plant CPA-FAS can be generated by any suitable method wellknown in the art, including but not limited to site-directedmutagenesis, randomized “point” mutagenesis, and domain-swap mutagenesisin which portions of the Sterculia CPA-FAS cDNA are “swapped” with theanalogous portion of other plant or bacterial CPA-FAS-encoding cDNAs(Back and Chappell (1996) PNAS 93: 6841-6845).

Variants may be produced by methods such as directed evolution or othertechniques for producing combinatorial libraries of variants. Thus, thepresent invention further contemplates a method of generating sets ofcombinatorial mutants of the present plant CPA-FAS proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (in other words, homologs) that possess the biologicalactivity of a CPA-FAS (for example, synthesis of CPA-FAs). In addition,screening such combinatorial libraries is used to generate, for example,novel plant CPA-FAS homologs that possess novel substrate specificitiesor other biological activities all together; examples of substratespecificities are described subsequently.

It is contemplated that the plant CPA-FAS nucleic acids (for example,SEQ ID NO: 1, and fragments and variants thereof) can be utilized asstarting nucleic acids for directed evolution. These techniques can beutilized to develop plant CPA-FAS variants having desirable propertiessuch as increased synthetic activity or altered affinity for aparticular fatty acid substrate.

In some embodiments, artificial evolution is performed by randommutagenesis (for example, by utilizing error-prone PCR to introducerandom mutations into a given coding sequence). This method requiresthat the frequency of mutation be finely tuned. As a general rule,beneficial mutations are rare, while deleterious mutations are common.This is because the combination of a deleterious mutation and abeneficial mutation often results in an inactive enzyme. The idealnumber of base substitutions for targeted gene is usually between 1.5and 5 (Moore and Arnold (1996) Nat. Biotech., 14, 458-67; Leung et al.(1989) Technique, 1:11-15; Eckert and Kunkel (1991) PCR Methods Appl.,1:17-24; Caldwell and Joyce (1992) PCR Methods Appl., 2:28-33; and Zhaoand Arnold (1997) Nuc. Acids. Res., 25:1307-08). After mutagenesis, theresulting clones are selected for desirable activity (for example,screened for CPA-FAS activity as described subsequently). Successiverounds of mutagenesis and selection are often necessary to developenzymes with desirable properties. It should be noted that only theuseful mutations are carried over to the next round of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (for example, Smith (1994) Nature, 370:324-25; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731). Gene shuffling involvesrandom fragmentation of several mutant DNAs followed by their reassemblyby PCR into full length molecules. Examples of various gene shufflingprocedures include, but are not limited to, assembly following DNasetreatment, the staggered extension process (STEP), and random priming invitro recombination. In the DNase mediated method, DNA segments isolatedfrom a pool of positive mutants are cleaved into random fragments withDNaseI and subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer (1994) Nature,370:398-91; Stemmer (1994) Proc. Natl. Acad. Sci. USA, 91, 10747-10751;Crameri et al. (1996) Nat. Biotech., 14:315-319; Zhang et al. (1997)Proc. Natl. Acad. Sci. USA, 94:4504-09; and Crameri et al. (1997) Nat.Biotech., 15:436-38). Variants produced by directed evolution can bescreened for CPA-FAS activity by the methods described subsequently (seeExample 3).

Homologs

Still other embodiments of the present invention provide isolatednucleic acid sequence encoding plant CPA-FAS homologs, and thepolypeptides encoded thereby. Some homologs of plant CPA-FAS haveintracellular half-lives dramatically different than the correspondingwild-type protein. For example, the altered protein are rendered eithermore stable or less stable to proteolytic degradation or other cellularprocess that result in destruction of, or otherwise inactivate plantCPA-FAS. Such homologs, and the genes that encode them, can be utilizedto alter the activity of plant CPA-FAS by modulating the half-life ofthe protein. For instance, a short half-life can give rise to moretransient plant CPA-FAS biological effects. Other homologs havecharacteristics which are either similar to wild-type plant CPA-FAS, orwhich differ in one or more respects from wild-type plant CPA-FAS.

The cDNA deduced amino acid sequence of Sterculia CPA-FAS is compared tothe cDNA deduced amino acid sequences of other known bacterial CPA-FASor CPA-FAS-like proteins, as shown in FIG. 10. The proposed S-adenosylmethionine binding motif (amino acid residues 171-179, using the E. colinumbering, and amino residues 627-635 in the Sterculia enzyme) and thecatalytically important cysteine (amino acid residue 354, using the E.coli numbering, and amino residue 822 in the Sterculia enzyme) areconserved for all proteins. Accordingly, in some embodiments, thepresent invention provides a plant CPA-FAS comprising at least the aminoacid motif V-L-D-I-G-C-G-W-G (the S-adenosyl methionine binding motif,corresponding to amino acid residues 627-635 in the Sterculia enzyme),or the nucleic acid sequences corresponding thereto. In yet otherembodiments of the present invention, it is contemplated that nucleicacid sequences suspected of encoding a plant CPA-FAS homolog is screenedby comparing motifs. In some embodiments, the deduced amino acidsequence can be analyzed for the presence of the amino acid motifV-L-D-I-G-C-G-W-G (the S-adenosyl methionine binding motif,corresponding to amino acid residues 627-635 in the Sterculia enzyme).

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of plantCPA-FAS homologs are aligned, preferably to promote the highest homologypossible. Such a population of variants can include, for example, plantCPA-FAS homologs from one or more species, or plant CPA-FAS homologsfrom the same species but which differ due to mutation. Amino acids thatappear at each position of the aligned sequences are selected to createa degenerate set of combinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialplant CPA-FAS library is produced by way of a degenerate library ofgenes encoding a library of polypeptides that each include at least aportion of candidate plant CPA-FAS-protein sequences. For example, amixture of synthetic oligonucleotides is enzymatically ligated into genesequences such that the degenerate set of candidate plant CPA-FASsequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (for example, for phage display)containing the set of plant CPA-FAS sequences therein.

There are many ways by which the library of potential plant CPA-FAShomologs can be generated from a degenerate oligonucleotide sequence. Insome embodiments, chemical synthesis of a degenerate gene sequence iscarried out in an automatic DNA synthesizer, and the synthetic genes areligated into an appropriate gene for expression. The purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential plant CPA-FAS sequences.The synthesis of degenerate oligonucleotides is well known in the art(See for example, Narang (1983) Tetrahedron Lett., 39:3-9; Itakura etal. (1981) Recombinant DNA, in Walton (ed.), Proceedings of the 3rdCleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289;Itakura et al. (1984) Annu. Rev. Biochem., 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucl. Acid Res., 11:477). Suchtechniques have been employed in the directed evolution of otherproteins (See for example, Scott et al. (1980) Science, 249:386-390;Roberts et al. (1992) Proc. Natl. Acad. Sci. USA, 89:2429-2433; Devlinet al. (1990) Science, 249: 404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA, 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Truncation Mutants of Plant Cyclopropane Fatty Acid Synthase

In addition, the present invention provides isolated nucleic acidsequences encoding fragments of plant CPA-FAS (for example, truncationmutants), and the polypeptides encoded by such nucleic acid sequences.In preferred embodiments, the plant CPA-FAS fragment is biologicallyactive. As described above, Sterculia CPA-FAS is contemplated to be anatural fusion of two polypeptide fragments possessing differentcatalytic activities; these two fragments catalyze either the formationof CPA-FA (the carboxy terminus, or the fragment from about amino acids397 ∀ about 20 amino acids to the end) or ∀-oxidation or a similarreaction (the amino terminus, or the fragment from the beginning toabout amino acid 397 ∀ about 20 amino acids). Therefore, it iscontemplated that the Sterculia CPA-FAS can be truncated into a carboxyterminus fragment and an amino terminus fragment (although eachtruncation fragment might overlap the other by a number of amino acids),by methods well known. It is contemplated that the truncation sitebetween the two domains is in the region of amino acids 393-401. It isfurther contemplated that these separate fragments will possess theassigned catalytic activity.

In some embodiments of the present invention, when expression of aportion of a plant CPA-FAS protein is desired, it may be necessary toadd a start codon (ATG) to the oligonucleotide fragment containing thedesired sequence to be expressed. It is well known in the art that amethionine at the N-terminal position can be enzymatically cleaved bythe use of the enzyme methionine aminopeptidase (MAP). MAP has beencloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol.,169:751-757) and Salmonella typhimurium and its in vitro activity hasbeen demonstrated on recombinant proteins (Miller et al. (1990) Proc.Natl. Acad. Sci. USA, 84:2718-1722). Therefore, removal of an N-terminalmethionine, if desired, can be achieved either in vivo by expressingsuch recombinant polypeptides in a host that produces MAP (for example,E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP.

Fusion Proteins Containing Plant Cyclopropane Fatty Acid Synthase

The present invention also provides nucleic acid sequences encodingfusion proteins incorporating all or part of plant CPA-FAS, and thepolypeptides encoded by such nucleic acid sequences. In someembodiments, the fusion proteins have a plant CPA-FAS functional domainwith a fusion partner. Accordingly, in some embodiments of the presentinvention, the coding sequences for the polypeptide (for example, aplant CPA-FAS functional domain) is incorporated as a part of a fusiongene including a nucleotide sequence encoding a different polypeptide.In one embodiment, a single fusion product polypeptide converts anunsaturated fatty acid to a CPA-FA (one fusion partner possesses theability to synthesize CPA-FA). In another embodiment, a single fusionproduct polypeptide converts an unsaturated fatty acid to a CPA-FA of aneven carbon number (one fusion partner possesses the ability tosynthesize CPA-FA, and a second fusion partner possess the ability toremove a single carbon from the CPA-FA). In yet another embodiment, asingle fusion product polypeptide converts an unsaturated fatty acid toa CPE-FA (one fusion partner possesses the ability to synthesize CPA-FA,and a second fusion partner possess the ability to desaturate theCPA-FA). In still another embodiment, a single fusion productpolypeptide converts an unsaturated fatty acid to a CPE-FA of an evencarbon number (one fusion partner possesses the ability to synthesizeCPA-FA, a second fusion partner possess the ability to desaturate theCPA-FA, and a third fusion partner possess the ability to remove asingle carbon from the CPA-FA).

In some embodiments of the present invention, chimeric constructs codefor fusion proteins containing a portion of a plant CPA-FAS and aportion of another gene. In some embodiments, the fusion proteins havebiological activity similar to the wild type plant CPA-FAS (for example,have at least one desired biological activity of plant CPA-FAS). Inother embodiments, the fusion protein have altered biological activity.

In other embodiments of the present invention, chimeric constructs codefor fusion proteins containing a plant CPA-FAS gene or portion thereofand a leader or other signal sequences which direct the protein totargeted subcellular locations. Such sequences are well known in theart, and direct proteins to locations such as the chloroplast, themitochondria, the endoplasmic reticulum, the tonoplast, the golginetwork, and the plasmalemma.

In addition to utilizing fusion proteins to alter biological activity,it is widely appreciated that fusion proteins can also facilitate theexpression and/or purification of proteins, such as a plant CPA-FASprotein of the present invention. Accordingly, in some embodiments ofthe present invention, a plant CPA-FAS is generated as aglutathione-S-transferase (in other words, GST fusion protein). It iscontemplated that such GST fusion proteins enables easy purification ofa plant CPA-FAS, such as by the use of glutathione-derivatized matrices(See for example, Ausabel et al. (eds.) (1991) Current Protocols inMolecular Biology, John Wiley & Sons, NY).

In another embodiment of the present invention, a fusion gene coding fora purification leader sequence, such as a poly-(His)/enterokinasecleavage site sequence at the N-terminus of the desired portion of aplant CPA-FAS allows purification of the expressed plant CPA-FAS fusionprotein by affinity chromatography using a Ni²⁺ metal resin. In stillanother embodiment of the present invention, the purification leadersequence is then subsequently removed by treatment with enterokinase(See for example, Hochuli et al. (1987) J. Chromatogr., 411:177; andJanknecht et al. Proc. Natl. Acad. Sci. USA, 88:8972). In yet otherembodiments of the present invention, a fusion gene coding for apurification sequence appended to either the N (amino) or the C(carboxy) terminus allows for affinity purification; one example isaddition of a hexahistidine tag to the carboxy terminus of a plantCPA-FAS which was optimal for affinity purification.

Techniques for making fusion genes are well known. Essentially, thejoining of various nucleic acid fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment of the present invention, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, in other embodiments of the presentinvention, PCR amplification of gene fragments is carried out usinganchor primers that give rise to complementary overhangs between twoconsecutive gene fragments that can subsequently be annealed to generatea chimeric gene sequence (See for example, Current Protocols inMolecular Biology, supra).

Screening Gene Products

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques are generally adaptable for rapid screening of the genelibraries generated by the combinatorial mutagenesis of plant CPA-FAShomologs. The most widely used techniques for screening large genelibraries typically comprise cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate sequences created by combinatorial mutagenesistechniques.

Accordingly, in one embodiment of the present invention, the candidateplant CPA-FAS gene products are displayed on the surface of a cell orviral particle, and the ability of particular cells or viral particlesto synthesize CPA-FAs is assayed using the techniques described in theExamples. In other embodiments of the present invention, the genelibrary is cloned into the gene for a surface membrane protein of abacterial cell, and the resulting fusion protein detected by panning (WO88/06630; Fuchs et al. (1991) BioTechnol., 9:1370-1371; and Goward etal. (1992) TIBS 18:136-140). In other embodiments of the presentinvention, fluorescently labeled molecules that bind plant CPA-FAS canbe used to score for potentially functional plant CPA-FAS homologs.Cells are visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In an alternate embodiment of the present invention, the gene library isexpressed as a fusion protein on the surface of a viral particle. Forexample, foreign peptide sequences are expressed on the surface ofinfectious phage in the filamentous phage system, thereby conferring twosignificant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of infection. The group of almostidentical E. coli filamentous phages M13, fd, and fl are most often usedin phage display libraries, as either of the phage gIII or gVIII coatproteins can be used to generate fusion proteins without disrupting theultimate packaging of the viral particle (See for example, WO 90/02909;WO 92/09690; Marks et al. (1992) J. Biol. Chem., 267:16007-16010;Griffths et al. (1993) EMBO J., 12:725-734; Clackson et al. (1991)Nature, 352:624-628; and Barbas et al. (1992) Proc. Natl. Acad. Sci.,89:4457-4461).

In another embodiment of the present invention, the recombinant phageantibody system (for example, RPAS, Pharmacia Catalog number 27-9400-01)is modified for use in expressing and screening of plant CPA-FAScombinatorial libraries. The pCANTAB 5 phagemid of the RPAS kit containsthe gene that encodes the phage gIII coat protein. In some embodimentsof the present invention, the plant CPA-FAS combinatorial gene libraryis cloned into the phagemid adjacent to the gIII signal sequence suchthat it is expressed as a gIII fusion protein. In other embodiments ofthe present invention, the phagemid is used to transform competent E.coli TGI cells after ligation. In still other embodiments of the presentinvention, transformed cells are subsequently infected with M13KO7helper phage to rescue the phagemid and its candidate plant CPA-FAS geneinsert. The resulting recombinant phage contain phagemid DNA encoding aspecific candidate plant CPA-FAS-protein and display one or more copiesof the corresponding fusion coat protein. In some embodiments of thepresent invention, the phage-displayed candidate proteins that arecapable of, for example, metabolizing a hydroperoxide, are selected orenriched by panning. The bound phage is then isolated, and if therecombinant phage express at least one copy of the wild type gIII coatprotein, they will retain their ability to infect E. coli. Thus,successive rounds of reinfection of E. coli and panning will greatlyenrich for plant CPA-FAS homologs, which can then be screened forfurther biological activities in order to differentiate agonists andantagonists.

In light of the present disclosure, other forms of mutagenesis generallyapplicable will be apparent to those skilled in the art in addition tothe aforementioned rational mutagenesis based on conserved versusnon-conserved residues. For example, plant CPA-FAS homologs can begenerated and screened using, for example, alanine scanning mutagenesisand the like (Ruf et al. (1994) Biochem., 33:1565-1572; Wang et al.(1994) J. Biol. Chem., 269:3095-3099; Balint (1993) Gene 137:109-118;Grodberg et al. (1993) Eur. J. Biochem., 218:597-601; Nagashima et al.(1993) J. Biol. Chem., 268:2888-2892; Lowman et al. (1991) Biochem.,30:10832-10838; and Cunningham et al. (1989) Science, 244:1081-1085), bylinker scanning mutagenesis (Gustin et al. (1993) Virol., 193:653-660;Brown et al. (1992) Mol. Cell. Biol., 12:2644-2652; McKnight et al.Science, 232:316); or by saturation mutagenesis (Meyers et al. (1986)Science, 232:613).

IV. Expression of Cloned Plant Cyclopropane Fatty Acid Synthase

In other embodiment of the present invention, nucleic acid sequencescorresponding to the plant CPA-FAS genes, homologs and mutants asdescribed above may be used to generate recombinant DNA molecules thatdirect the expression of the encoded protein product in appropriate hostcells.

As will be understood by those of skill in the art, it may beadvantageous to produce plant CPA-FAS-encoding nucleotide sequencespossessing non-naturally occurring codons. Therefore, in some preferredembodiments, codons preferred by a particular prokaryotic or eukaryotichost (Murray et al. (1989) Nucl. Acids Res., 17) can be selected, forexample, to increase the rate of plant CPA-FAS expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from naturally occurringsequence.

A. Vectors for Production of Plant Cyclopropane Fatty Acid Synthase

The nucleic acid sequences of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thenucleic acid sequence may be included in any one of a variety ofexpression vectors for expressing a polypeptide. In some embodiments ofthe present invention, vectors include, but are not limited to,chromosomal, nonchromosomal and synthetic DNA sequences (for example,derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral DNA such as vaccinia, adenovirus, fowl pox virus, andpseudorabies). It is contemplated that any vector may be used as long asit is replicable and viable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the nucleic sequencesas broadly described above (for example, SEQ ID NO:1). In someembodiments of the present invention, the constructs comprise a vector,such as a plasmid or viral vector, into which a nucleic acid sequence ofthe invention has been inserted, in a forward or reverse orientation. Inpreferred embodiments of the present invention, the appropriate nucleicacid sequence is inserted into the vector using any of a variety ofprocedures. In general, the nucleic acid sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44,PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Anyother plasmid or vector may be used as long as they are replicable andviable in the host. In some preferred embodiments of the presentinvention, plant expression vectors comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. In other embodiments, DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

In certain embodiments of the present invention, a nucleic acid sequenceof the present invention within an expression vector is operativelylinked to an appropriate expression control sequence(s) (promoter) todirect mRNA synthesis. Promoters useful in the present inventioninclude, but are not limited to, the LTR or SV40 promoter, the E. colilac or trp, the phage lambda P_(L) and P_(R), T3 and T7 promoters, andthe cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)thymidine kinase, and mouse metallothionein-I promoters and otherpromoters known to control expression of gene in prokaryotic oreukaryotic cells or their viruses. In other embodiments of the presentinvention, recombinant expression vectors include origins of replicationand selectable markers permitting transformation of the host cell (forexample, dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or tetracycline or ampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding polypeptides of the present invention by higher eukaryotes isincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp that acton a promoter to increase its transcription. Enhancers useful in thepresent invention include, but are not limited to, the SV40 enhancer onthe late side of the replication origin bp 100 to 270, a cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

B. Host Cells for Production of Plant Cyclopropane Fatty Acid Synthase

In a further embodiment, the present invention provides host cellscontaining any of the above-described constructs. In some embodiments ofthe present invention, the host cell is a higher eukaryotic cell (forexample, a plant cell). In other embodiments of the present invention,the host cell is a lower eukaryotic cell (for example, a yeast cell). Instill other embodiments of the present invention, the host cell can be aprokaryotic cell (for example, a bacterial cell). Specific examples ofhost cells include, but are not limited to, Escherichia coli, Salmonellatyphimurium, Bacillus subtilis, and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, as well as Saccharomycescerivisiae, Schizosaccharomycees pombe, Drosophila S2 cells, SpodopteraSf9 cells, Chinese hamster ovary (CHO) cells, COS-7 lines of monkeykidney fibroblasts, (Gluzman (1981) Cell 23:175), 293T, C127, 3T3, HeLaand BHK cell lines, NT-1 (tobacco cell culture line), root cell andcultured roots in rhizosecretion (Gleba et al. (1999) Proc Natl Acad SciUSA 96: 5973-5977). Other examples include microspore-derived culturesof oilseed rape. (Weselake R J and Taylor D C (1999) Prog. Lipid Res.38: 401), and transformation of pollen and microspore culture systems.Further examples are described in the Examples.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by any of the recombinant sequences ofthe present invention described above. In some embodiments, introductionof the construct into the host cell can be accomplished by calciumphosphate transfection, DEAE-Dextran mediated transfection, orelectroporation (See for example, Davis et al. (1986) Basic Methods inMolecular Biology). Alternatively, in some embodiments of the presentinvention, a polypeptide of the invention can be synthetically producedby conventional peptide synthesizers.

Proteins can be expressed in eukaryotic cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom a DNA construct of the present invention. Appropriate cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y.

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (for example, temperature shift or chemical induction)and cells are cultured for an additional period. In other embodiments ofthe present invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

VI. Production of Large Quantities of Cyclopropane Fatty Acids

In one aspect of the present invention, methods are provided forproducing large quantities of CPA-FAs. In some embodiments, CPA-FAs areproduced in vivo, in organisms transformed with a heterologous geneencoding a polypeptide exhibiting plant cyclopropane fatty acid synthaseactivity and grown under conditions sufficient to effect production ofCPA-FAs. In other embodiments, CPA-FAs are produced in vitro, fromeither nucleic acid sequences encoding a plant CPA-FAS or frompolypeptides exhibiting plant cyclopropane fatty acid synthase activity.

A. In Vivo in Transgenic Organism

In some embodiments of the present invention, CPA-FAs are produced invivo, by providing an organism transformed with a heterologous geneencoding a plant CPA-FAS activity and growing the transgenic organismunder conditions sufficient to effect production of CPA-FAs. In otherembodiments of the present invention, CPA-FAs are produced in vivo bytransforming an organism with a heterologous gene encoding a plantCPA-FAS and growing the transgenic organism under conditions sufficientto effect production of CPA-FAs. Illustrative examples of transgenicorganisms are provided in the Examples.

Organisms which are transformed with a heterologous gene encoding aplant CPA-FAS include preferably those which naturally synthesize andstore in some manner fatty acids, and those which are commerciallyfeasible to grow and suitable for harvesting large amounts of the fattyacid products. Such organisms include but are not limited to bacteria,oleaginous yeast and algae, and plants. Examples of bacteria include E.coli and related bacteria which can be grown in commercial-scalefermenters. Examples of plants include preferably oil-producing plants,such as soybean, rapeseed and canola, sunflower, cotton, corn, cocoa,safflower, oil palm, coconut palm, flax, castor, and peanut. Manycommercial cultivars can be transformed with heterologous genes. Incases where that is not possible, non-commercial cultivars of plants canbe transformed, and the trait for expression of plant CPA-FAS moved tocommercial cultivars by breeding techniques well-known in the art.

A heterologous gene encoding a plant CPA-FAS, which includes mutants orvariants of a plant CPA-FAS, includes any suitable sequence of theinvention as described above. Preferably, the heterologous gene isprovided within an expression vector such that transformation with thevector results in expression of the polypeptide; suitable vectors aredescribed above and following.

A transgenic organism is grown under conditions sufficient to effectproduction of CPA-FAs. In some embodiments of the present invention, atransgenic organism is supplied with exogenous substrates of the plantCPA-FAS (as for example as in a fermenter). Such substrates compriseunsaturated fatty acids; the number of double bonds is from one to morethan one, and the chain length of such unsaturated fatty acids isvariable, but is preferably about 14 to 22 carbons in length. The fattyacid substrate may also comprise additional functional groups, includingbut not limited to acetylenic bonds, conjugated acetylenic and ethylenicbonds, allenic groups, furan rings, and epoxy-, and keto-groups; two ormore of these functional groups may be found in a single fatty acid. Thesubstrates are either free fatty acids, or their salts. Substrates maybe supplied in various forms as are well known in the art; such formsinclude aqueous suspensions prepared by sonication, aqueous suspensionsprepared with detergents and other surfactants, dissolution of thesubstrate into a solvent, and dried powders of substrates. Such formsmay be added to organisms or cultured cells or tissues grown infermenters.

In yet other embodiments of the present invention, a transgenic organismcomprises a heterologous gene encoding a plant CPA-FAS operably linkedto an inducible promoter, and is grown either in the presence of the aninducing agent, or is grown and then exposed to an inducing agent. Instill other embodiments of the present invention, a transgenic organismcomprises a heterologous gene encoding a plant CPA-FAS operably linkedto a promoter which is either tissue specific or developmentallyspecific, and is grown to the point at which the tissue is developed orthe developmental stage at which the developmentally-specific promoteris activated. Such promoters include seed specific promoters.

In alternative embodiments, a transgenic organism as described above isengineered to produce greater amounts of the unsaturated substrate. Inone embodiment, a transgenic organism is co-transformed with aheterologous gene encoding a protein which desaturates fatty acids, suchthat the fatty acid desaturase is expressed. More preferably, the plantCPA-FAS and the heterologous fatty acid desaturase are targeted to thesame intracellular location; most preferably, such a location serves tosynthesize oil, such as a microsome in plants. These co-transformantsare then grown under conditions sufficient to effect production ofCPA-FAs. In some embodiments of the present invention, a co-transformantis supplied with exogenous substrates of the fatty acid desaturase; suchsubstrates comprise saturated and unsaturated fatty acids. The chainlength of such unsaturated fatty acids is variable, but is preferablyabout 14 to 22 carbons in length. The fatty acid substrate may alsocomprise additional functional groups, including but not limited toacetylenic bonds, conjugated acetylenic and ethylenic bonds, allenicgroups, and epoxy-, and keto-groups; two or more of these functionalgroups may be found in a single fatty acid. The substrates are eitherfree fatty acids, or are fatty acids incorporated into a largermolecule, such as a glycerolipid. Most preferably, the fatty acidsubstrate is esterified to a phospholipid. Substrates may be supplied,added, or applied as described above.

In other embodiments, the heterologous genes are under control ofpromoters which are either inducible, tissue-specific, ordevelopmentally specific, and the organism is grown as described above,such that the heterologous genes encoding polypeptides with the fattyacid desaturase and the plant CPA-FAS activities are expressed.

In yet further embodiments of the invention, an organism is transformedwith a nucleotide sequence coding for a fusion protein comprising both afatty acid desaturase and a plant CPA-FAS, as described above, such thatboth enzymatic activities are expressed. Such transgenic organisms aregrown as described above.

In other embodiments of the present invention, a host organism is onewhich produces large amounts of the substrate. For example, it iscontemplated that oleate is a preferred substrate of Sterculia CPA-FA;thus, a particularly suitable host in one which produces a highproportion of oleic acid. Such hosts include plant lines bred to producehigh oleic oils, such as sunflower or corn; such lines are produced fromindividual plants in which FAD2 of naturally low levels is selected, aswell as those plants subjected to mutagenesis and subsequently selectedfor decreased FAD2 activity, and those plants subjected to knock-outtechnology, in which FAD2 is silenced by antisense or co-suppression. Inother lines, the synthesis of shorter chain fatty acids is alsodecreased, by any of the means described above, resulting in increasedexpression of oleic acid. Any of these modifications may also becombined, to produce plant lines with even higher amounts of oleic acid.

In other embodiments of the present invention, the methods for producinglarge quantities of CPA-FAs further comprise collecting the CPA-FAsproduced. Such methods are known generally in the art, and includeharvesting the transgenic organisms and extracting the CPA-FAs (see, forexample, Christie, W. W. (1982) Lipid Analysis, 2^(nd) Edition (PergamonPress, Oxford); and Kates, M (1986) Techniques of Lipidology (Elsevier,Amsterdam)). Extraction procedures preferably include solventextraction, and typically include disrupting cells, as by chopping,mincing, grinding, and/or sonicating, prior to solvent extraction. Inone embodiment, lipids are extracted from the tissue according to themethod of Bligh and Dyer (1959) (Can J Biochem Physiol 37: 911-917);fatty acids esterified to glycerolipids can be hydrolyzed under acidicor alkaline conditions and collected by solvent extraction. In yet otherembodiments of the present invention, the CPA-FAs are further purified,as for example by thin layer liquid chromatography, gas-liquidchromatography, or high pressure liquid chromatography.

1. Transgenic Plants, Seeds, and Plant Parts

Plants are transformed with a heterologous gene encoding a plant CPA-FASor co-transformed with a first heterologous gene encoding plant CPA-FASand with a second heterologous gene encoding fatty acid desaturase ortransformed with a fusion gene encoding a fusion polypeptide expressinga plant CPA-FAS and fatty acid desaturase activities according toprocedures well known in the art. It is contemplated that theheterologous genes are utilized to increase the level of the enzymeactivities encoded by the heterologous genes.

a. Plants

The methods of the present invention are not limited to any particularplant. Indeed, a variety of plants are contemplated, including but notlimited to tomato, potato, tobacco, pepper, rice, corn, barley, wheat,Brassica, Arabidopsis, sunflower, soybean, poplar, and pine. Preferredplants include oil-producing species, which are plant species whichproduce and store triacylglycerol in specific organs, primarily inseeds. Such species include but are not limited to soybean (Glycinemax), rapeseed and canola (including Brassica napus and B. campestris),sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zeamays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oilpalm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linumusitatissimum), castor (Ricinus communis) and peanut (Arachis hypogaea).The group also includes non-agronomic species which are useful indeveloping appropriate expression vectors such as tobacco, rapid cyclingBrassica species, and Arabidopsis thaliana, and wild species which maybe a source of unique fatty acids. Preferred plant lines include thosewhich are high monounsaturates, and in particular high oleates, asdescribed previously. Particularly preferred plant lines are oilseedcrop lines with a high oleate background. The high oleate background canbe generated, for example, by using existing plant lines which arederived through breeding and/or mutagenesis (for example, high oleicsunflower lines and high oleate rapeseed lines), or by using geneticallyengineered lines, such as the high oleate soybean generated by fad2co-suppression, or the OLE1 gene to reduce saturates

b. Vectors

The methods of the present invention contemplate the use of aheterologous gene encoding a plant CPA-FAS, as described above. Themethods of the present invention further contemplate the use of a secondheterologous gene which encodes a fatty acid desaturase; suchpolypeptides are known (See, for example, Polashock J J, Chin C-K,Martin C E (1992) Plant Physiol. 100: 894-901, which describesexpression of the yeast delta-9 fatty acid desaturase in Nicotianatabaccum. The yeast delta-9 fatty acid desaturase is the OLE1 gene,which can increase 16:1 and 18:1 levels in plant tissues.) Heterologousgenes encoding mutants and variants of fatty acid desaturases areprepared as described above for plant CPA-FAS. Heterologous genesencoding a fusion CPA-FAS/fatty acid desaturase is prepared as describedabove.

Heterologous genes intended for expression in plants are first assembledin expression cassettes comprising a promoter. Methods which are wellknown to those skilled in the art may be used to construct expressionvectors containing a heterologous gene and appropriate transcriptionaland translational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are widely described in the art (See forexample, Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al.(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y).

In general, these vectors comprise a nucleic acid sequence of theinvention encoding a plant CPA-FAS (as described above) operably linkedto a promoter and other regulatory sequences (for example, enhancers,polyadenylation signals, etc.) required for expression in a plant.

Promoters include but are not limited to constitutive promoters,tissue-, organ-, and developmentally-specific promoters, and induciblepromoters. Examples of promoters include but are not limited to:constitutive promoter 35S of cauliflower mosaic virus; a wound-induciblepromoter from tomato, leucine amino peptidase (“LAP,” Chao et al. (1999)Plant Physiol 120: 979-992); a chemically-inducible promoter fromtobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH(benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomatoproteinase inhibitor II promoter (PIN2) or LAP promoter (both induciblewith methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267);a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); andseed-specific promoters, such as those for seed storage proteins (forexample, phaseolin, napin, oleosin, and a promoter for soybean betaconglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). All referencescited herein are incorporated in their entirety.

The expression cassettes may further comprise any sequences required forexpression of mRNA. Such sequences include, but are not limited totranscription terminators, enhancers such as introns, viral sequences,and sequences intended for the targeting of the gene product to specificorganelles and cell compartments.

A variety of transcriptional terminators are available for use inexpression of sequences using the promoters of the present invention.Transcriptional terminators are responsible for the termination oftranscription beyond the transcript and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants include, but are not limited to, the CaMV 35Sterminator, the tm1 terminator, the pea rbcS E9 terminator, and thenopaline and octopine synthase terminator (See for example, Odell et al.(1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineauet al. (1991) Mol. Gen. Genet., 262:141; Proudfoot (1991) Cell, 64:671;Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell,2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) NucleicAcids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

In addition, in some embodiments, constructs for expression of the geneof interest include one or more of sequences found to enhance geneexpression from within the transcriptional unit. These sequences can beused in conjunction with the nucleic acid sequence of interest toincrease expression in plants. Various intron sequences have been shownto enhance expression, particularly in monocotyledonous cells. Forexample, the introns of the maize AdhI gene have been found tosignificantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells (Calais et al. (1987)Genes Develop. 1: 1183). Intron sequences have been routinelyincorporated into plant transformation vectors, typically within thenon-translated leader.

In some embodiments of the present invention, the construct forexpression of the nucleic acid sequence of interest also includes aregulator such as a nuclear localization signal (Calderone et al. (1984)Cell 39:499; Lassoer et al. (1991) Plant Molecular Biology 17:229), aplant translational consensus sequence (Joshi (1987) Nucleic AcidsResearch 15:6643), an intron (Luehrsen and Walbot (1991) Mol. Gen.Genet. 225:81), and the like, operably linked to the nucleic acidsequence encoding plant CPA-FAS.

In preparing the construct comprising a nucleic acid sequence encodingplant CPA-FAS, various DNA fragments can be manipulated, so as toprovide for the DNA sequences in the desired orientation (for example,sense or antisense) orientation and, as appropriate, in the desiredreading frame. For example, adapters or linkers can be employed to jointhe DNA fragments or other manipulations can be used to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resection, ligation, or the likeis preferably employed, where insertions, deletions or substitutions(for example, transitions and transversions) are involved.

Numerous transformation vectors are available for plant transformation.The selection of a vector for use will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers are preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing and Vierra (1982) Gene 19:259; Bevan et al. (1983) Nature 304:184), the bar gene which confersresistance to the herbicide phosphinothricin (White et al. (1990) NuclAcids Res. 18: 1062; Spencer et al. (1990) Theor. Appl. Genet. 79: 625),the hph gene which confers resistance to the antibiotic hygromycin(Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4:2929), and thedhfr gene, which confers resistance to methotrexate (Bourouis et al.(1983) EMBO J., 2:1099).

In some preferred embodiments, the vector is adapted for use in anAgrobacterium mediated transfection process (See for example, U.S. Pat.Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all ofwhich are incorporated herein by reference). Construction of recombinantTi and Ri plasmids in general follows methods typically used with themore common bacterial vectors, such as pBR322. Additional use can bemade of accessory genetic elements sometimes found with the nativeplasmids and sometimes constructed from foreign sequences. These mayinclude but are not limited to structural genes for antibioticresistance as selection genes.

There are two systems of recombinant Ti and Ri plasmid vector systemsnow in use. The first system is called the “cointegrate” system. In thissystem, the shuttle vector containing the gene of interest is insertedby genetic recombination into a non-oncogenic Ti plasmid that containsboth the cis-acting and trans-acting elements required for planttransformation as, for example, in the pMLJ1 shuttle vector and thenon-oncogenic Ti plasmid pGV3850. The second system is called the“binary” system in which two plasmids are used; the gene of interest isinserted into a shuttle vector containing the cis-acting elementsrequired for plant transformation. The other necessary functions areprovided in trans by the non-oncogenic Ti plasmid as exemplified by thepBIN19 shuttle vector and the non-oncogenic Ti plasmid PALA404. Some ofthese vectors are commercially available.

In other embodiments of the invention, the nucleic acid sequence ofinterest is targeted to a particular locus on the plant genome.Site-directed integration of the nucleic acid sequence of interest intothe plant cell genome may be achieved by, for example, homologousrecombination using Agrobacterium-derived sequences. Generally, plantcells are incubated with a strain of Agrobacterium which contains atargeting vector in which sequences that are homologous to a DNAsequence inside the target locus are flanked by Agrobacteriumtransfer-DNA 1-DNA) sequences, as previously described (U.S. Pat. No.5,501,967). One of skill in the art knows that homologous recombinationmay be achieved using targeting vectors which contain sequences that arehomologous to any part of the targeted plant gene, whether belonging tothe regulatory elements of the gene, or the coding regions of the gene.Homologous recombination may be achieved at any region of a plant geneso long as the nucleic acid sequence of regions flanking the site to betargeted is known.

In yet other embodiments, the nucleic acids of the present invention isutilized to construct vectors derived from plant (+) RNA viruses (forexample, brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus,cucumber mosaic virus, tomato mosaic virus, and combinations and hybridsthereof). Generally, the inserted plant CPA-FAS polynucleotide of thepresent invention can be expressed from these vectors as a fusionprotein (for example, coat protein fusion protein) or from its ownsubgenomic promoter or other promoter. Methods for the construction anduse of such viruses are described in U.S. Pat. Nos. 5,846,795;5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of whichare incorporated herein by reference.

In some embodiments of the present invention, where the nucleic acidsequence of interest is introduced directly into a plant. One vectoruseful for direct gene transfer techniques in combination with selectionby the herbicide Basta (or phosphinothricin) is a modified version ofthe plasmid pCIB246, with a CaMV 35S promoter in operational fusion tothe E. coli GUS gene and the CaMV 35S transcriptional terminator (WO93/07278).

c. Transformation Techniques

Once a nucleic acid sequence encoding a plant CPA-FAS is operativelylinked to an appropriate promoter and inserted into a suitable vectorfor the particular transformation technique utilized (for example, oneof the vectors described above), the recombinant DNA described above canbe introduced into the plant cell in a number of art-recognized ways.Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant targeted for transformation. In someembodiments, the vector is maintained episomally. In other embodiments,the vector is integrated into the genome.

In some embodiments, direct transformation in the plastid genome is usedto introduce the vector into the plant cell (See for example, U.S. Pat.Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783). Thebasic technique for chloroplast transformation involves introducingregions of cloned plastid DNA flanking a selectable marker together withthe nucleic acid encoding the RNA sequences of interest into a suitabletarget tissue (for example, using biolistics or protoplasttransformation with calcium chloride or PEG). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate homologous recombinationwith the plastid genome and thus allow the replacement or modificationof specific regions of the plastome. Initially, point mutations in thechloroplast 16S rRNA and rps12 genes conferring resistance tospectinomycin and/or streptomycin are utilized as selectable markers fortransformation (Svab et al. (1990) PNAS, 87:8526; Staub and Maliga,(1992) Plant Cell, 4:39). The presence of cloning sites between thesemarkers allowed creation of a plastid targeting vector introduction offoreign DNA molecules (Staub and Maliga (1993) EMBO J., 12:601).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab and Maliga (1993) PNAS,90:913). Other selectable markers useful for plastid transformation areknown in the art and encompassed within the scope of the presentinvention. Plants homoplasmic for plastid genomes containing the twonucleic acid sequences separated by a promoter of the present inventionare obtained, and are preferentially capable of high expression of theRNAs encoded by the DNA molecule.

In other embodiments, vectors useful in the practice of the presentinvention are microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA (Crossway(1985) Mol. Gen. Genet, 202:179). In still other embodiments, the vectoris transferred into the plant cell by using polyethylene glycol (Krenset al. (1982) Nature, 296:72; Crossway et al. (1986) BioTechniques,4:320); fusion of protoplasts with other entities, either minicells,cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al.(1982) Proc. Natl. Acad. Sci., USA, 79:1859); protoplast transformation(EP 0 292 435); direct gene transfer (Paszkowski et al. (1984) EMBO J.,3:2717; Hayashimoto et al. (1990) Plant Physiol. 93:857).

In still further embodiments, the vector may also be introduced into theplant cells by electroporation. (Fromm, et al. (1985) Pro. Natl Acad.Sci. USA 82:5824; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602). In this technique, plant protoplasts are electroporated in thepresence of plasmids containing the gene construct. Electrical impulsesof high field strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

In yet other embodiments, the vector is introduced through ballisticparticle acceleration using devices (for example, available fromAgracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.). (Seefor example, U.S. Pat. No. 4,945,050; and McCabe et al. (1988)Biotechnology 6:923). See also, Weissinger et al. (1988) Annual Rev.Genet. 22:421; Sanford et al. (1987) Particulate Science and Technology,5:27 (onion); Svab et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8526(tobacco chloroplast); Christou et al. (1988) Plant Physiol., 87:671(soybean); McCabe et al. (1988) Bio/Technology 6:923 (soybean); Klein etal. (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al.(1988) Bio/Technology, 6:559 (maize); Klein et al. (1988) PlantPhysiol., 91:4404 (maize); Fromm et al. (1990) Bio/Technology, 8:833;and Gordon-Kamm et al. (1990) Plant Cell, 2:603 (maize); Koziel et al.(1993) Biotechnology, 11:194 (maize); Hill et al. (1995) Euphytica,85:119 and Koziel et al. (1996) Annals of the New York Academy ofSciences 792:164; Shimamoto et al. (1989) Nature 338: 274 (rice);Christou et al. (1991) Biotechnology, 9:957 (rice); Datta et al. (1990)Bio/Technology 8:736 (rice); European Patent Application EP 0 332 581(orchardgrass and other Pooideae); Vasil et al. (1993) Biotechnology,11: 1553 (wheat); Weeks et al. (1993) Plant Physiol., 102: 1077 (wheat);Wan et al. (1994) Plant Physiol. 104: 37 (barley); Jahne et al. (1994)Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller (1991) Planta,185:330 (barley); Umbeck et al. (1987) Bio/Technology 5: 263 (cotton);Casas et al (1993) Proc. Natl. Acad. Sci. USA 90:11212 (sorghum); Somerset al. (1992) Bio/Technology 10:1589 (oat); Torbert et al. (1995) PlantCell Reports, 14:635 (oat); Weeks et al. (1993) Plant Physiol., 102:1077(wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al. (1994) ThePlant Journal, 5:285 (wheat).

In addition to direct transformation, in some embodiments, the vectorscomprising a nucleic acid sequence encoding a plant CPA-FAS of thepresent invention are transferred using Agrobacterium-mediatedtransformation (Hinchee et al. (1988) Biotechnology, 6:915; Ishida etal. (1996) Nature Biotechnology 14:745). Agrobacterium is arepresentative genus of the gram-negative family Rhizobiaceae. Itsspecies are responsible for plant tumors such as crown gall and hairyroot disease. In the dedifferentiated tissue characteristic of thetumors, amino acid derivatives known as opines are produced andcatabolized. The bacterial genes responsible for expression of opinesare a convenient source of control elements for chimeric expressioncassettes. Heterologous genetic sequences (for example, nucleic acidsequences operatively linked to a promoter of the present invention),can be introduced into appropriate plant cells, by means of the Tiplasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted toplant cells on infection by Agrobacterium tumefaciens, and is stablyintegrated into the plant genome (Schell (1987) Science, 237: 1176).Species which are susceptible infection by Agrobacterium may betransformed in vitro. Alternatively, plants may be transformed in vivo,such as by transformation of a whole plant by Agrobacteria infiltrationof adult plants, as in a “floral dip” method (Bechtold N, Ellis J,Pelletier G (1993) Cr. Acad. Sci. III-Vie 316:1194-1199).

d. Regeneration

After selecting for transformed plant material which can express theheterologous gene encoding a plant CPA-FAS, whole plants areregenerated. Plant regeneration from cultured protoplasts is describedin Evans et al. (1983) Handbook of Plant Cell Cultures, Vol. 1:(MacMillan Publishing Co. New York); and Vasil I. R. (ed.), Cell Cultureand Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I(1984), and Vol. III (1986). It is known that many plants can beregenerated from cultured cells or tissues, including but not limited toall major species of sugarcane, sugar beet, cotton, fruit and othertrees, legumes and vegetables, and monocots (for example, the plantsdescribed above). Means for regeneration vary from species to species ofplants, but generally a suspension of transformed protoplasts containingcopies of the heterologous gene is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.

Alternatively, embryo formation can be induced from the protoplastsuspension. These embryos geminate and form mature plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. The reproducibility of regenerationdepends on the control of these variables.

e. Generation of Transgenic Lines

Transgenic lines are established from transgenic plants by tissueculture propagation. The presence of nucleic acid sequences encodingexogenous a plant CPA-FAS of the present invention (including mutants orvariants thereof may be transferred to related varieties by traditionalplant breeding techniques.

These transgenic lines are then utilized for evaluation of oilproduction and other agronomic traits.

B. In Vitro Systems

In other embodiments of the present invention, CPA-FAs are produced invitro, from either nucleic acid sequences encoding a plant CPA-FAS orfrom polypeptides exhibiting plant cyclopropane fatty acid synthaseactivity.

1. Using Nucleic Acid Sequences Encoding Plant Cyclopropane Fatty AcidSynthase

In some embodiments of the present invention, methods for producinglarge quantities of CPA-FAs comprise adding an isolated nucleic acidsequence encoding a plant CPA-FAS to in vitro expression systems underconditions sufficient to cause production of CPA-FAs. The isolatednucleic acid sequences encoding a plant cyclopropane is any suitablesequence of the invention as described above, and preferably is providedwithin an expression vector such that addition of the vector to an invitro transcription/translation system results in expression of thepolypeptide. The system further comprises the substrates for plantCPA-FAS, as previously described. Alternatively, the system furthercomprises the means for generating the substrates for plant CPA-FAS.Such means include but are not limited to the provision of at least oneprotein exhibiting fatty acid desaturase activity, and substrates forfatty acid desaturase, as described above.

In other embodiments of the present invention, the methods for producinglarge quantities of CPA-FAs further comprise collecting the CPA-FAsproduced. Such methods are known generally in the art. In yet otherembodiments of the present invention, the CPA-FAs are further purified,as for example by thin layer liquid chromatography, gas-liquidchromatography, or high pressure liquid chromatography.

2. Using Plant Cyclopropane Synthase Polypeptides

In some embodiments of the present invention, methods for producinglarge quantities of CPA-FAs comprise incubating a plant CPA-FAS underconditions sufficient to result in the synthesis of CPA-FAs; generally,such incubation is carried out in a mixture which comprises the plantCPA-FAS.

A plant CPA-FAS, as described previously, is obtained by purification ofeither naturally occurring plant CPA-FAS or recombinant plant CPA-FASfrom an organism transformed with heterologous gene encoding a plantCPA-FAS, as previously described. A source of naturally occurring plantCPA-FAS is contemplated to include but not limited to plants, as forexample Malvaceae, Sterculiaceae, Bombaceae, Tilaceae, Mimosaceae andSapindaceae. A source of recombinant plant CPA-FAS is either plant,bacterial or other transgenic organisms, transformed with heterologousgene encoding plant CPA-FAS as described above. The recombinant plantCPA-FAS may include means for improving purification, as for example a6x-His tag added to the C-terminus of the protein as described above.Alternatively, plant CPA-FAS is chemically synthesized.

The incubation mixture further comprises the substrates for plantCPA-FAS, as described above. Alternatively, the mixture furthercomprises the means for generating the substrates for plant CPA-FAS.Such means include but are not limited to the provision of at least oneprotein exhibiting fatty acid desaturase activity, and appropriatesubstrates for fatty acid desaturase, as described above. Additionalsubstrates include but are not limited substrates for the synthesis ofphospholipids, such as lyso-phospholipid and phospholipidacyl-transferase, as well as phospholipid liposomes with lipid transferproteins; particularly preferred phospholipids are phosphatidylcholines.

In other embodiments of the present invention, the methods for producinglarge quantities of CPA-FAs further comprise collecting the CPA-FAsproduced; such methods are described above.

VII. Production of Cyclopropane Fatty Acids Where Not Normally Present

In another aspect of the present invention, methods are provided forproducing CPA-FAs in organisms and/or tissues where CPA-FAs are notusually present or are present in very low levels. In this aspect,CPA-FAs are produced in organisms transformed with a heterologous geneencoding a polypeptide exhibiting plant cyclopropane fatty acid synthaseactivity and grown under conditions sufficient to effect production ofCPA-FAs. In some embodiments, the methods comprise production of CPA-FAsin specific tissues or organs, such as in plant roots. In otherembodiments, the methods comprise production of CPA-FAs at specificdevelopmental phases. In yet other embodiments, the methods compriseproduction of CPA-FAs in specific tissues or organs and at specificdevelopmental phases.

In this aspect, the CPA-FAs are contemplated to serve a physiologicalrole. For example, it is contemplated that CPA-FAs provide fungalresistance to plant roots. Thus, expression of CPA-FAS in plant rootswhich normally do not possess CPA-FAs, or possess CPA-FAs atinsignificant levels, provides increased fungal resistance.

In some embodiments of the present invention, the methods compriseproviding a transgenic organism comprising a heterologous gene encodinga plant CPA-FAS operably linked to an inducible promoter, and growingthe transgenic organism either in the presence of the an inducing agent,or growing the organism and then exposing it to an inducing agent,thereby expressing CPA-FAS resulting in the production of CPA-FAs. Instill other embodiments of the present invention, the methods compriseproviding a transgenic organism comprising a heterologous gene encodinga plant CPA-FAS operably linked to a promoter which is either tissuespecific or developmentally specific, and growing the transgenicorganism to the point at which the tissue is developed or thedevelopmental stage at which the developmentally-specific promoter isactivated, thereby expressing CPA-FAS resulting in the production ofCPA-FAs. Exemplary promoters include but are not limited to seedspecific promoters.

A heterologous gene encoding a plant CPA-FAS, which includes mutants orvariants of a plant CPA-FAS, includes any suitable sequence of theinvention as described above. Preferably, the heterologous gene isprovided within an expression vector such that transformation with thevector results in expression of the polypeptide; suitable vectors aredescribed above and following.

Methods of producing transgenic organisms, and in particular transgenicplants, are described above.

VIII. Manipulation of Plant Cyclopropane Fatty Acid Synthase Activity inPlants

As noted above, CPE-FAs are considered an anti-nutritional factor infood oils, yet many seed lipids containing CPE-FAs are extensivelyconsumed by humans, especially in tropical areas, and by animals as well(for example, cottonseed meal is a typical animal feed product, and is aby-product of cottonseed processing to obtain the oil). Because of thesehealth concerns, vegetable oils containing CPE-FAs must be treated withhigh temperature or hydrogenation before consumption. These treatmentsadd to the oil processing costs, and also result in the presence of acertain percentage of trans fatty acids produced due the hydrogenation;the presence of such trans fatty acids are also undesirable. Therefore,the present invention provides methods to eliminate CPE-FAs from seedoils, as well as plants which produce oils with reduced levels ofCPE-FAs, and oils with reduced levels CPE-FAs which are not treated withhigh temperature or hydrogenation to reduce CPE-FAs levels and whichhave low levels of trans fatty acids. These aspects of the inventionhave great utility in significantly reducing oil processing costs,decreasing the presence of undesirable hydrogenated fatty acids, andenhancing the value of both the seed oils, of unprocessed seed and ofprocessed seed meal for food consumption. Some embodiments of thepresent invention provides methods to decrease the amount of CPE-FAsfrom plant seed oils by cyclopropane synthase gene silencing technology.Other embodiments provide methods of decreasing the amount of CPE-FAsfrom cotton seed oils by cyclopropane synthase gene silencing technology

It is further contemplated that the nucleic acids encoding a plantCPA-FAS of the present invention may be utilized to either increase ordecrease the level of plant CPA-FAS mRNA and/or protein in transfectedcells as compared to the levels in wild-type cells. Such transgeniccells have great utility, including but not limited to further researchas to the effects of the overexpression of plant CPA-FAS, and as to theeffects as to the underexpression or lack of plant CPA-FAS.

Accordingly, in some embodiments, expression in plants by the methodsdescribed above leads to the overexpression of plant CPA-FAS intransgenic plants, plant tissues, or plant cells.

In other embodiments of the present invention, the plant CPA-FASpolynucleotides are utilized to decrease the level of plant CPA-FASprotein or mRNA in transgenic plants, plant tissues, or plant cells ascompared to wild-type plants, plant tissues, or plant cells. One methodof reducing plant CPA-FAS expression utilizes expression of antisensetranscripts. Antisense RNA has been used to inhibit plant target genesin a tissue-specific manner (for example, van der Krol et al. (1988)Biotechniques 6:958-976). Antisense inhibition has been shown using theentire cDNA sequence as well as a partial cDNA sequence (for example,Sheehy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8805-8809; Cannon etal. (1990) Plant Mol. Biol. 15:3947). There is also evidence that 3′non-coding sequence fragment and 5′ coding sequence fragments,containing as few as 41 base-pairs of a 1.87 kb cDNA, can play importantroles in antisense inhibition (Ch'ng et al. (1989) Proc. Natl. Acad.Sci. USA 86:10006-10010).

Accordingly, in some embodiments, a plant CPA-FAS encoding-nucleic acidof the present invention (for example, SEQ ID NO: 1, and fragments andvariants thereof) are oriented in a vector and expressed so as toproduce antisense transcripts. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the antisense strand of RNA will be transcribed. Theexpression cassette is then transformed into plants and the antisensestrand of RNA is produced. The nucleic acid segment to be introducedgenerally will be substantially identical to at least a portion of theendogenous gene or genes to be repressed. The sequence, however, neednot be perfectly identical to inhibit expression. The vectors of thepresent invention can be designed such that the inhibitory effectapplies to other proteins within a family of genes exhibiting homologyor substantial homology to the target gene.

Furthermore, for antisense suppression, the introduced sequence alsoneed not be full length relative to either the primary transcriptionproduct or fully processed mRNA. Generally, higher homology can be usedto compensate for the use of a shorter sequence. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andhomology of non-coding segments may be equally effective. Normally, asequence of between about 30 or 40 nucleotides and about full lengthnucleotides should be used, though a sequence of at least about 100nucleotides is preferred, a sequence of at least about 200 nucleotidesis more preferred, and a sequence of at least about 500 nucleotides isespecially preferred.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of the target gene or genes. It is possible to designribozymes that specifically pair with virtually any target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules, making it a true enzyme. The inclusion ofribozyme sequences within antisense RNAs confers RNA-cleaving activityupon them, thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, Solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff, et al. (1988) Nature 334:585-591. Ribozymestargeted to the mRNA of a lipid biosynthetic gene, resulting in aheritable increase of the target enzyme substrate, have also beendescribed (Merlo A O et al. (1998) Plant Cell 10: 1603-1621).

Another method of reducing CPA-FAS expression utilizes the phenomenon ofcosuppression or gene silencing (See for example, U.S. Pat. No.6,063,947, incorporated herein by reference). The phenomenon ofcosuppression has also been used to inhibit plant target genes in atissue-specific manner. Cosuppression of an endogenous gene using afull-length cDNA sequence as well as a partial cDNA sequence (730 bp ofa 1770 bp cDNA) are known (for example, Napoli et al. (1990) Plant Cell2:279-289; van der Krol et al. (1990) Plant Cell 2:291-299; Smith et al.(1990) Mol. Gen. Genetics 224:477-481). Accordingly, in some embodimentsthe nucleic acid sequences encoding a plant CPA-FAS of the presentinvention (for example including SEQ ID NOs: 1, and fragments andvariants thereof) are expressed in another species of plant to effectcosuppression of a homologous gene.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For cosuppression, the introduced sequence in the expression cassette,needing less than absolute identity, also need not be full length,relative to either the primary transcription product or fully processedmRNA. This may be preferred to avoid concurrent production of someplants which are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Normally, a sequence of the size ranges noted above forantisense regulation is used.

An effective method to down regulate a gene is by hairpin RNAconstructs.

Guidance to the design of such constructs for efficient, effective andhigh throughput gene silencing have been described (Wesley S V et al.(2001) Plant J. 27: 581-590). Another method to decrease expression of agene (either endogenous or exogenous) is via siRNAs. siRNAs can beapplied to a plant and taken up by plant cells; alternatively, siRNAscan be expressed in vivo from an expression cassette.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); :m(micromolar); mol (moles); mmol (millimoles); mol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); :g(micrograms); ng (nanograms); l or L (liters); ml (milliliters); :l(microliters); cm (centimeters); mm (millimeters); :m (micrometers); nm(nanometers); ° C. (degrees Centigrade); PCR (polymerase chainreaction); RT-PCR (reverse-transcriptase-PCR); TAIL-PCR (thermalasymmetric interlaced-PCR); EST, expressed sequence tag; BLAST; FAME,fatty acid methyl ester; GC/MS, gas chromatography/mass spectrometry;TLC, thin layer chromatography; SC medium; NT medium; MES; 2,4-D;CPA-FA, cyclopropane fatty acids; CPE-FA, cyclopropene fatty acids;DHSA, dihydrosterculic acid; SCPA-FAS, Sterculia cyclopropane fatty acidsynthase; MSU, Michigan State University;

EXAMPLE 1 Experimental Procedures

Materials

Developing Seeds

Sterculia foetida developing seeds were collected from Jul. 20 to Oct.15, 1998 at Miami Montgomery Botanical Center (Florida USA 331564242).Upon receipt of the fresh seeds, the seed coats were removed and thecotyledons and embryos were either immediately used fresh for labelingexperiments or frozen and stored at −80° C. for subsequent RNAextraction and lipids analysis.

Tobacco Suspension Cells

Tobacco suspension cells (Nicotiana tabaccum L. cv. Bright yellow 2)were maintained in liquid medium containing Murashige and Skoog basalsalts (Gibco, Grand island, NY), 3% sucrose, 2.5 mM MES/KOH pH 5.7, 1mg/ml thiamine, 1 mg/ml myo-inositol, and 1:M 2,4-D. Cultures weresub-cultured weekly with 5% (v/v) inoculum from a 7-day old culture andshaken at 28° C. in 200 ml flasks.

Chemicals

Radio-isotopes, L-[methyl-¹⁴C] methionine (55 mCi/mmol), [1-¹⁴C] acetate(55 mCi/mmol) and [1-¹⁴C] oleic acid (50 mCi/mmol) were purchased fromAmerican Radiolabeled Chemicals, Inc.).

Lipid Analysis

To determine the fatty acid accumulation during Sterculia seeddevelopment, 2 mg triacylglycerol (13:0-1.89 mg) were added as aninternal standard to 200 mg seed tissue (fresh weight) before the lipidextraction. Lipids were extracted at each developmental stage accordingto the method of Bligh and Dyer (1959) Can J Biochem Physiol 37,911-917). Fatty acid methyl esters (FAMES) were prepared by transferringan aliquot of the lipid extract into a new tube and drying the extractunder nitrogen. 2.5 ml of 1% sodium methoxide in methanol and 2.5 ml ofheptane were added to each tube, and the mixture vortexed at roomtemperature for 2 to 3 minutes. 2.5 ml of water was added to each tube,and the mixture vortexed briefly. The FAMES were extracted by washingthe mixture three times with 2.5 ml of hexane, and the combined organicphases were then washed two times with 3.0 ml of water, and dried undernitrogen. The FAMES were then analyzed by GC-MS, using a Hewlett Packard5890 gas chromatography configured with an autosampler and HP MSD 5972mass analyzer (quadruple, operating in electron impact mode). Separationof the FAMEs was carried out on a DB 23 column of 30 m long withdiameter of 0.25 mm.

Lipid extraction, preparation of fatty acid methyl esters, and theiranalysis by GC/MS for yeast and tobacco suspension cells followed thesame procedure as described above for Sterculia seeds, except that nointernal fatty acid standard was added. In order to accurately determinethe identity of dihydrosterculic acid (DHSA) in transgenic tobaccosuspension cells, the saturated and unsaturated fatty acid methyl esterswere separated by argentation TLC (Norris et al. (1967) J Chromatography31: 69-76). Argentation plates (15% silver nitrate) were developedsequentially at −20° C. to heights of 8, 13, and 19 cm in toluene. Thefatty acid methyl esters on the plates were located by spraying with0.2% (w/v) 2′,7′-Dichlorofluorescein in ethanol. The saturated fattyacid methyl-ester bands on top of the argentation plates were scrapedinto test tubes, recovered by elution with 6 ml of hexane:ethyl ether(2:1, v/v), and analyzed by GC/MS.

Assay of Cyclopropane Fatty Acid Synthesis

Cyclopropane fatty acid synthase in tissue homogenates was assayed in areaction mixture containing 0.1 ml of cell free homogenate, 0.02-0.05 mMoleoyl-CoA, and 0.02 mM [¹⁴C-methyl]S-adenosylmethionine substrate, in atotal volume of 0.2 ml. The presence of oleoyl-CoA enhances activityabout two-fold over its absence, but higher concentrations of oleoyl-CoAare inhibitory. The Km for S-adenosylmethionine in the crude extract is0.02 mM. The assay cocktail was incubated at 30° C. for 1 hour. Theassay was terminated by the addition of 0.5 ml of aqueous KOH and 1.0 mlof ethanol, and allowed to stand overnight to give completesaponification of the lipids. On acidification, the labeled free fattyacids were extracted into hexane, then the hexane phase washed withwater and evaporated to dryness. An aliquot of the organic phase wasassayed for radioactivity. The remainder of the product was analyzed byTLC. A portion of the fatty acid products may also be derivatized withethereal diazomethane.

Radiolabeling Sterculia Developing Seeds and Transgenic Tobacco Cells

Developing seeds from the collection of August 25 were used for labelingexperiments. One cotyledon was sliced into 8 pieces, incubated in 0.5 mlof labeling buffer (50 mM phosphate pH 7.0 with 5:Ci of radio-isotope)at room temperature shaking constantly. The labeling was terminated at30, 60, and 120 minutes by immediate lipid extraction. The fatty acidmethyl-esters from these samples were separated on C18 reverse phase TLCin the solvent system of acetonitrile:methanol:water (75:25:0.5, v/v).The radioactivity was visualized with Instant-Imager.

Independent transgenic callus was transferred back into liquid mediumand the cells sub-cultured as described above. After three days ofsubculture, 5:Ci of L-[methyl-¹⁴C] methionine or [1-¹⁴C] oleic acid wasadded to the medium, and the cells were incubated for an additional 24hours. The cells were then collected by brief centrifugation, followedby lipid extraction and preparation of fatty acid methyl-esters.Saturated fatty acids were separated from unsaturated fatty acids byargentation TLC, the individual saturated fatty acids were separatedfrom each other by C18 reverse phase TLC, and the radio-active spotswere recovered for GC/MS analysis.

Library Construction and Sequencing

Equal amounts of developing seeds from the seeds collected on August 5,August 25, September 10, and September 30 were pooled together. Duringthis period of time, the deposition of oil showed a linear increase (seeExample 2). Ten grams of the pooled developing seeds were ground into afine powder in liquid nitrogen. The RNA was extracted as described bySchultz et al (1994). The quality of the isolated total RNA was analyzedby separation on 1% formaldehyde agarose gel. The cDNA library wasprepared from the total isolated RNA of Sterculia developing seeds byStratagene (11011 North Torrey Pines Road, La Jolla, Calif. 92037). ThecDNAs were directionally cloned into Uni-ZAP® at EcoRI and XhoI sites.Mass excisions were performed according the protocol provided byStratagene. A total of 21,120 clones were picked and grown in 220'96-well plates. Twenty-eight out the 220 plates were directly sequencedat the MSU sequencing facility. The remaining 192 plates (18432 clones)were spotted on filters by Genome System for library subtraction. Basedon the information obtained from the first 1,500 sequences from thenon-subtracted library, three most abundant sequences were chosen tosubtract from the library on the filters. A total of seventy 96-wellplates were re-racked and sequenced.

Constructs For Tobacco and Yeast Expression

A putative Sterculia cyclopropane synthase (SCPA-FAS) gene wasidentified based on sequence homology with bacterial cyclopropanesynthase. The complete cDNA of the putative (SCPA-FAS) is 2977 bp longand was compiled from two overlapping clones R50-D5 (1-1732 bp) andC15-C3 (933-2977 bp). Both clones were sequenced from both strands. Inorder to put the complete putative SCPA-FAS together, the sequenceencoding amino acids 1-335 was amplified from clone R50-D5 with primersJO886 (TCCTCTAGACTCGAGCCCGGGATGGGAGTGGCT GTGATCGGTGGTGGGATC) and JO883(GTTGTAAGACGTCGTGTAACTCGGTC ATACAATTCG), and the sequence encoding aminoacids 336-864 and containing the stop codon was amplified from cloneC15-C3 with primers JO884 (CAATGTGCTGCAGAATGTTGGGAAAACAAGTCAGCC) andJO885 (GGGAGATCTCGAGCCTATTTACTTTTFGATAAAGTTAATAGGC). For ease ofcloning, a null mutation was made at the third position of the codon foramino acid 335 lysine, which changed the lysine codon from CTA to CTG,so that a Pst I site could be created. The upstream fragment wasinserted into pBluescript KS at XhoI and PstI sites, and the downstreamfragment was inserted at PstI and XbaI. The resulting construct wasnamed pBluescript KS-SCPAS; this construct was re-sequenced from bothstrands, and found to be error-free. The complete SCPA-FAS was releasedfrom pbluescript KS at SmaI and XbaI sites and inserted into the binaryvector pE1776 between SmaI and XbaI site. pE1776 carries theconstitutive promoter “Super Promoter” and an ags terminator. Theresulting construct was named as pE1776-SCPAS and transferred intoAgrobacterium strain LBA4404 for tobacco suspension cell transformation.

For the yeast construct, in order to create an EcoRI site upstream,amino acid 1-335 was amplified from clone R50-D5 with primer JO968(GCCCTCGAGAATTCTAAAATGTCTG TGGCTGTGATCGGTGGTGGGATCCAAGG GCTGG) andJO883R (GTTGTAAGAC GTCGTGTAACTCGGTCATACAATTCG). The PCR fragment wasdigested with XhoI and PstI, and used to replace the correspondingsection in the construct of pBluescript KS-SCPAS. The SCPA-FAS wasreleased with EcoRI and XbaI, and inserted into the yeast vectorpYES2/CT (Invitrogen). The resulting construct was named aspYES2/CT-SCPAS

Expression of SCPA-FAS in Yeast and Tobacco Suspension Cells

The construct pYES2/CT-SCPAS was transformed into yeast strain InvSc1using the S.c.EasyComp™ Transformation Kit (Invitrogen Cat# K5050-01).To express SCPA-FAS, a yeast colony containing pYES2/CT-SCPAS wasinoculated into 50 ml of SC medium with 2% galactose and 200 mg/L oleicacid. The culture was grown at 28° C. shaking at 150 rpm. After 48 hrgrowth, yeast cells were collected by brief centrifugation; the yeastlipids extracted were extracted from the pellet, and fatty acidmethyl-esters prepared from the extracted lipids and analyzed by GC/MS.

Agrobacterium mediated tobacco transformation was carried out asdescribed by Rempel and Nelson (1991). The agrobacterial culture wasgrown overnight, and 100 ul of culture containing the proper constructwas added to 4 ml samples of 3-day-old tobacco suspension cells. Thecells were then cultured at 28° C. for three days, and then pelleted bybrief centrifugation and washed three more times with NT mediumcontaining 100 ug/ml kanamycin and 500 ug/ml carbenicillin. The washedcells were spread on selection plates (NT medium with addition 0.7%phytagar, 100 mg/L kanamycin, and 500 mg/L carbenicillin). After threeto four weeks, independent transformants were transferred to new plates.Once enough tissue was collected, the lipids were extracted and thefatty acid composition analyzed by GC/MS.

EXAMPLE 2 Identification of Cyclopropane Synthase from Sterculia foetidaDeveloping Seeds Assay of Cyclopropane Fatty Acid Synthase

Frozen endosperm tissue, harvested from developing seeds of Sterculiafoetida and stored at −70° C., was ground to a fine powder in liquidnitrogen. One weight of powder was thawed in two volumes of buffercontaining 0.1 M Na tricine, pH 7.0, 1% w/v defatted BSA, 1% w/v PVP40,15% v/v glycerol and 1 mM 2-mercaptoethanol. The slurry was brieflyhomogenized and filtered through miracloth. The filtrate was stored onice. The residual paste was re-homogenized in two volumes of the abovebuffer, refiltered and the filtrate combined with the first filtrate.The combined filtrate, designated as “cell free homogenate” can be usedfor enzyme assay or subsequently fractionated. The use of tricine buffer(compared to tris or phosphate buffer), the use of pH 7.0 (compared topH 6.4 or 7.8), and the addition of BSA and PVP40 (compared to noadditions) each enhanced activity recovered in the cell free homogenateby approximately two-fold.

The activity of cyclopropane fatty acid synthase was assayed asdescribed above. When analyzed without derivitization, labeled freefatty acid was the major constituent in the saponified product (>90%).When analyzed after derivitization with ethereal diazomethane, labeledfatty acid methyl ester was the major product (>90%). When the labeledfatty acid methyl esters were analyzed by C18 reversed-phase TLC therewas a single radioactive spot co-eluting with the methyldihydrosterculate standard. Thus the radioactivity recovered in thehexane phase after saponification is a good measure of the total labelin [14C-methyl]dihydrosterculate.

Maximum cyclopropane fatty acid synthase activities of the order of0.5-1 nmole/min/gram fresh weight of seed tissue were measured. Theactivity was susceptible to inhibition by a wide range of detergents(CHAPS, Triton-X100, octyl glucoside, sodium deoxycholate,cetylpyridinium chloride and lysophosphatidylcholine). In fractionationstudies most of the activity remained in the supernatant plus fat layerfraction after centrifugation at 10,000×g for 5 minutes. Subsequentcentrifugation at 100,000×g for one hour produced a microsomal pelletthat contained 72% of the total cyclopropane fatty acid synthaseactivity found in the cell free homogenate, but only 1.5% of the totalprotein, to give a specific activity enhancement of 48-fold. The actionof detergents and the behavior of activity during centrifugation areconsistent with the cyclopropane fatty acid synthase being either amembrane-associated or an integral membrane protein. When the reactionwith the cell free homogenate was terminated by lipid extraction, mostof the labeled dihydrosterculate was found in the phosphatidylcholinefraction. This suggests that oleoyl-phosphatidylcholine is a substratefor the enzyme.

Thus, the initial assay for cyclopropane fatty acid synthase andcharacterization of the activity indicates that developing seeds ofSterculia foetida are able to synthesize CPA-FA. Moreover, the enzymeappears to be a membrane-associated or integral membrane protein, andthe substrate appears to be oleoyl-phosphatidylcholine.

Lipid Deposition During Seed Development

Developing seeds from Sterculia foetida were collected at 7 time pointsspanning a period of 100 days, from Jul. 20 to Oct. 15, 1998, at theMiami Montgomery Botanical Center. Each pod contained 10 to 20 seeds.The pods were dark green and the seeds were white in July; as the seedsdeveloped, the color of the pod gradually turned red, while seeds turnedbrown. The cotyledons of the first collection, on July 20, were stillvery watery, and the seeds from the last collection, on October 28, werealmost completely dried. The fatty acids from all of the seedcollections, with the exception of the last collection in which the seedwere quite dry, were analyzed as described above.

The profile of fatty acid accumulation of the developing seeds is shownin FIG. 2. The results show that the total fatty acids and CPE-FAsaccumulated at a linear rate from August 5 to October 14. During thesame period of time, the percent of the CPE-FAs increased from 40% to60% of the total fatty acids. The fatty acid composition at 90 days wasvery similar to data reported for mature seeds (Bohannon and Kleiman(1978) Lipids 13(4): 270-273).

These results also suggested that if the original hypothesis is correct,that CPE-FAs are derived from CPA-FAs, developing seed of Sterculiafoetida have high activities of cyclopropane fatty acid synthase, andshould be a good source of mRNA encoding this enzyme.

Identification and Isolation of cDNA Encoding of Cyclopropane Synthase

In order to maximize the presence of cyclopropane synthase anddesaturase in the cDNA library, developing Sterculia foetida seeds atstages when CPE-FAs accumulate at the highest rate should be used toprepare a cDNA library. As shown in FIG. 2, CPE-FAs accumulated atessentially a linear rate from August 5 to October 14. Therefore, equalamount of developing seeds from the collections of August 5, August 25,September 10, September 30, and October 14 pooled together and used forRNA extraction.

The RNA was shipped to Stratagene for cDNA library construction. Theprimary plaques were 2.6×10⁷ pfu with average insert size of 1.7 kb.After mass excision, performed as described above, a total of 21,120clones were picked and grown in 220 96-well plates. Twenty-eight plateswere directly sequenced at the Michigan State University sequencingfacility. About 1,500 sequences were obtained with an average readinglength of 500 bp. Blast searches (Translated BLAST Searches: Nucleotidequery—Protein db [blastx]) at NCBI of these 1,500 un-subtractedsequences identified Legumin A, Legumin B, and a non-specific lipidtransfer protein as the three most abundant sequences; these sequenceswere selected for subsequent library subtraction. The remaining 192(18,432 clones) plates were spotted on filters by Genome System forlibrary subtraction. The three selected sequences represented about 30%of the clones on the filters. A total of seventy 96-well plates (withthe positive clones removed, or subtracted) were re-racked; all of these70 plates have been sequenced. Approximately 3,800 sequences with 500 bpwere obtained. Blast searches of these sequences were also performed.

Based on the blast results, 23 ESTs showed some level of similarity withbacterial cyclopropane synthase. After compiling these EST sequences, itwas very clear that all the 23 ESTs were derived from the same gene. Thedistribution of the ESTs along the gene is shown in FIG. 3. A fulllength clone was assembled, for which the nucleic acid sequence of thegene is shown in FIG. 4. The predicted putative cyclopropane synthase is864 amino acids long, as shown in FIG. 5. The bacterial cyclopropanesynthase is 382 amino acids long, which is less than half the size ofSterculia cyclopropane synthase. A comparison of the Sterculia enzyme tothe E. coli enzyme revealed that the Sterculia sequence is 49% similar(188/376) and 32% identical (122/376) to the E. coli sequence over theregion of overlap, which is the carboxy terminal (see FIG. 6). TheSterculia enzyme has an additional approximately 470 amino acids at theamino terminal.

EXAMPLE 3 Characterization of Cyclopropane Fatty Acid Synthase FromSterculia

Functional Analysis of the Putative SCPA-FAS in Yeast

Yeast cells possess several characteristics which make it a particularlyuseful test organism for evaluating whether the putative SCPA-FAS geneisolated from developing Sterculia seeds did in fact encode SCPA-FAS.These characteristics include the facts that yeast is a eucaryoticorganism and that its fatty acid composition is very simple and consistsonly of 16:0, 16:1, 18:0, and 18:1. Thus, if dihydrosterculic acid couldbe detected in yeast transfected with a gene encoding SCPA-FAS, it wouldconfirm that the SCPA-FAS is functioning as a cyclopropane synthase.

The yeast expression vector pYES2/CT was used, and the SCPA-FAS codingsequence was placed under the under the control of a galactose induciblepromoter, as described above. Yeast was transfected with the expressionvector and grown also as described above. Under the assumption thatoleic acid (18:1⁾⁹) is the most likely precursor of this enzyme, oleicacid was added to the medium to final concentration of 200 mg/L. Thefatty acids of 15 yeast colonies containing pYES2/CT-SCPAS and 5 controlcolonies with the pYES2/CT-LacZ were analyzed, and the results shown inFIG. 6.

As shown in FIG. 6 (A), oleic acid was effectively incorporated in yeastlipids.

Some contamination of the oleic acid used in the feeding experiments bylinolenic acid (18:2) was also apparent, as the presence of linolenicshowed up in the GC spectrum at 32.78 minutes. As shown in FIG. 6(B), atiny unique peak with a retention time 34.44 minutes was present in allthe 15 colonies of cells transfected with the SCPA-FAS gene but wasabsent in all the control samples. This peak was identified asdihydrosterculic fatty acid methyl ester based upon two lines ofevidences. First, the retention time of this peak is the same as that ofthe dihydrosterculic acid methyl ester standard. Second, the massspectrum of this peak was identical to the dihydrosterculic acid methylester standard as shown in FIG. 6(C). Therefore, the putative SCPA-FASenzyme from Sterculia does function as cyclopropane synthase.

Functional Analysis of the Putative SCPA-FAS Tobacco Suspension Cells

Although the function of SCPA-FAS was confirmed in yeast system, it wasimportant to evaluate its function in plant tissues. Tobacco suspensioncells (Bright Yellow 2) possess several characteristics particularlyuseful as a test plant system for evaluating the function of theSCPA-FAS gene isolated from developing Sterculia seeds in transgenicplant cells. The cell line is well characterized cell line and veryeasily to transformed. The suspension cells don't contain any CPA-FAs,and provide sufficient tissue for lipid analysis within 40 days of aftertransformation. Of even greater importance, it has been well documentedthat tobacco callus are more tolerant to unusual fatty acids.

The SCPA-FAS-encoding nucleic acid was transformed into tobaccosuspension cells under the control of a constitutive promoter; at thesame time, an empty vector was also transformed into tobacco cells ascontrol. After the transformation, independent transformants weretransferred to new plates and subcultured with 20-day intervals. Lipidswere extracted from 15 test transformants with pE1776-SCAPS, and from 12control transformants with the empty vector pE1776. The lipids wereextracted and the fatty acid methyl-esters prepared from these samplesas described above. Saturated FAMEs were separated from other FAMEs withargentation TLC; then the saturated FAMEs were analyzed by GC/MS alongwith methyl-ester of DHSA standard. The results are shown in FIG. 7. Themajor saturated fatty acids in control tobacco callus transformed withthe empty vector of pE1776 were 16:0, 18:0, and some 20:0, as is shownin FIG. 7(A). In test tobacco callus transformed with putative Sterculiacyclopropane synthase (SCPA-FAS), the same fatty acids were present aswere in the control samples, with an additional prominent peak withretention time of 35.69 minutes, as is shown in FIG. 7(B). Thisadditional peak was identified as dihydrosterculic acid by comparison toa dihydrosterculic acid standard, which had the same retention time asthe additional peak, 35.69 minutes, as is shown in FIG. 7(C). To confirmits identity, the additional peak was further analyzed by massspectrometry; the results are shown in FIG. 8. The mass spectrum ofcontrol dihydrosterculic acid is characterized by be a molecular ion of310 along with other unique ions like 278 (M-32) and 236 (M-74), asshown in FIG. 8(B). The mass spectrum of the additional peak with aretention time of 35.69 minutes, as shown in FIG. 8(D), was nearlyidentical to that of the dihydrosterculic acid standard. Therefore,based upon both the retention time and mass spectra comparisons, theadditional fatty acid found in tobacco callus transformed with nucleicacid encoding Sterculia CPA-FAS was identified as dihydrosterculic acid.The fatty acid compositions of 15 independent transformants with nucleicacid encoding SCPA-FAS were analyzed, and the dihydrosterculic acidcontents in these transformants ranged from 3% to 6%, as shown in FIG.9. The average amount of dihydrosterculic acid present was approximately4%.

Elucidation of the Cyclopropene Ring Formation

The pathway for the synthesis of sterculic acid proposed by Yano et al.((1972) Lipids 7: 35-45), based upon radioisotope labeling experiments,was an initial formation of dihydrosterculic acid from oleic acid, withsubsequent of desaturation dihydrosterculic acid to sterculic acid. Yanoet al further suggested that the ring methylene group was derived fromthe methyl group of methionine. This proposal was confirmed bypreliminary labeling studies with Sterculia developing seeds. Whendeveloping seeds were labeled with ¹⁴C-ethionine, the majority ofradioactivity was first found in DHSA, with small amounts resent insterculic acid; with longer periods of incubation, more radioactivityaccumulated in the sterculic acid.

Additional labeling experiments were carried out with tobacco suspensioncells transformed with either nucleic acid encoding SCPA-FAS (SCPAS-2and SPAS-11, two independent transgenic lines transformed withpE1776-SCPAS) or an the empty vector. The suspension cells wereincubated with either [1-¹⁴C] oleic acid or L-[methyl-¹⁴C] methioninefor 24 hours. The FAMEs from the labeled cells were then separated byargentation TLC and the distribution of radioactivity was visualizedwith Instant Imager. When incubated with [1-₁₄C] oleic acid, the testtransformants carrying nucleic acid encoding SCPA-FAS yielded about 3.5%of the total radioactivity in the top band containing only saturatedfatty acids, whereas control transformants carrying the empty vectorpE1776 resulted in no radioactivity associated with saturated fattyacids. These results demonstrate that the presence of SCPA-FAS resultedin the conversion of oleic acid (18:1) into saturated fatty acid, whichwas most likely dihydrosterculic acid. Moreover, the majority of [1-¹⁴C]oleic acid taken up was further desaturated to linoleic (18:2). Whencells were incubated with L-[methyl-1-¹⁴C] methionine, radioactivity wasfound in the saturated fatty acid band only from the cells transformedwith nucleic acid encoding SCPA-FAS, but not in the control cells. Fromthe previous analyses of the saturated fatty acids of both the test andcontrol transformants, the only difference between the two was that DHSAwas found exclusively in transformants with nucleic acid encodingSCPA-FAS. Therefore, the radioactivity associated with the saturatedfatty acids is most likely DHSA.

To confirm that the radioactive fatty acid was in fact DHSA, thesaturated FAMEs obtained from pE1776-SCPAS-2 after incubation witheither [1-₁₄C] oleic acid or L-[methyl-¹⁴C] methionine were eluted fromthe argentation plate, and the individual saturated FAMEs were separatedby C18 reverse phase TLC. The radioactive FAMEs labeled from either[1-¹⁴C] oleic acid or L-[methyl-¹⁴C] methionine were located at the sameposition as the DHSA standard. The radioactive spots were then elutedfrom the C18 plate and analyzed by GC/MS. The radioactive FAMEs weretherefore identified as DHSA based upon both the retention time and themass spectra.

Taken together, these results demonstrate that SCPA-FAS synthesizesdihydrosterculic acid by transferring a methylene group fromS-adenosyl-methionine to oleic acid.

EXAMPLE 4 Preparation Of Antibody Against Sterculia CyclopropaneSynthase

Antibody was prepared to Sterculia cyclopropane fatty acid synthase(CPA-FAS) expressed in a bacterial expression system. The conservedcarboxyl terminal region (about 390 amino acids) of the protein waschosen for antibody production, and was amplified by PCR and expressedin E. coli with a HIS tag. An alignment between the Sterculia and thebacterial CPA-FAS is shown in FIG. 1 1; the portion of the SterculiaCPA-FAS from about amino acid 470 to amino acid 864 was used to prepareantibody.

The vector utilized was pET28a(+) (Novagen, inc., 601 Science Drive,Madison, Wis. 53711), where the site of insertion is EcoR I and Sac I.

The PCR primers used to amplify the partial Sterculia cyclopropanesynthase were as follows: JO873 CCGGAATTCTGTTCTCTTAAAACAGCTCTGAAAGTGCJO885 CCCTCTAGAGCTGGGATAAATGAAAACTATTTCAATTATCCG

The location of primers in the Sterculia cyclopropane synthase are shownin FIG. 12. PCR reactions were performed under the following conditions:Template [R15-C3]  5.0 ul Buffer [10 X] 10.0 ul dNTP [10 mM]  2.0 ulJO873 [4.1 pM/ul]  9.0 ul JO885 [7.3 pM/ul]  5.0 ul Water 68.5 ul PWO 0.5 ul 94° C.  5 min 94° C. 30 seconds 55° C. 30 seconds 72° C. 30seconds 30 Cycles72° C. 7 min

Three separate samples were subjected to PCR, designated EA-I, EA-II,and EA-III. After the reactions, the PCR fragments were purified with aQiagen PCR purification kit.

The PCR fragments and the vector were then digested with the restrictionenzymes EcoRI and SacI. The PCR fragments were inserted into the vectorsby ligation overnight at 4° C. with T4 DNA ligase, at an insert:vectorratio of 4:1. The resulting constructs, designated pET28a-EA-I,pET28a-EA-II, and pET28a-EA-III, were transformed into DH5∀. Thecolonies were screened to identify the correct constructs; spin-prepswere prepared from pET28a-EA-II-4 and pET28a-EA-III-5. These twoconstructs were transformed into BL21 for protein expression. Theexpressed protein is shown in FIG. 13, where the portion of the aminoacid sequence highlighted is derived from the vector, which contains a6-histidine tag for purification.

The expression of the partial sterculia CPA-FAS was then induced asfollows: BL21 containing pET28a-EA-114 or pET28a-EA-III-5 wereinoculated into 2 ml of LB with 50 ug/ml kanamycin, and grown at 37° C.overnight with shaking. The next morning, 1.5 ml of the overnightculture was inoculated into 500 ml LB with 50 ug/ml kanamycin; the cellswere grown for 2.5 hours. IPTG was then added to a final concentrationof 0.5 mM. The cells were grown for an additional 5 hours, and collectedby centrifuging at 5,000 g for 10 min. The collected cells were eitherstored at −20° C. or used directly for protein extraction.

Protein was extracted from collected cells as follows: Cells from 500 mlculture were resuspended into 60 ml of cell lysis buffer (20 mMTris-HCl, pH7.4; 0.2 mM NaCl; 10 mMB-mercaptoethanol; 1 mM Benzamidine;1% (v/v) Triton X-100; and 1 mM PMSF). The cells were disrupted byultrasonic treatment using three 20-second pulses at 50 watts on ice,and the suspension centrifuged at 10,000 g for 20 min. The solublefraction was the supernatant, and the insoluble fraction contained theinclusion bodies.

Inclusion bodies were purified from the insoluble fraction as follows:The insoluble fraction was resuspended in 8 ml of buffer (50 mMTris-HCl, pH 8.0; 1 mM EDTA; 25% (w/v) Sucrose; and 25 mg Lysozyme).MgCl₂, MnCl₂, and DNase I were then added to final concentrations 10 mM,1 mM, and 10 ug/ml, respectively. The mixture was incubated at roomtemperature for 30 min, and 20 ml of the following buffer was added (0.2M NaCl; 1% Deoxycholic acid; 1.6% (v/v) Triton X-100; 20 mM Tris-HCl,pH7.5; and 2 mM EDTA). The mixture was centrifuged at 5000×g for 10minutes, and the pellet resuspended in 0.5% triton X-100/1 mM EDTA andrecentrifuged. This procedure was repeated until a tight pellet wasobtained.

The inclusion bodies were then subjected to 10% SDS PAGE, and thepredominant band of the partial Sterculia CPA-FAS was cut out and storedat −20° C. The frozen gel slices were sent to Cocalico Biologicals, Inc.(Reamstown, Pa.) for antibody preparation in rabbits.

EXAMPLE 5 Identification and Characterization of Cyclopropane Fatty AcidSynthase from Cotton

Identification of Sequences Coding Cotton CPA-FAS

The amino acid sequence of Sterculia CPA-FAS was used to blast NCBI ESTdatabase. Three ESTs were found from cotton. All three are from the samelibrary (fibers isolated from bolls harvested 7-10 dpa) and weresequenced at Clemson University Genomics Institute. These ESTs aredesignated and described as follows, and are shown in FIG. 14.

EST1

-   -   gi|13351381|gb|BG441729.1|BG441729    -   GA_Ea0014H01f7-10 dpa fiber library    -   Gossypium arboreum cDNA clone GA_Ea0014H01f.

EST2

-   -   gi|21094588|gb|BQ406901.1|BQ406901    -   GA_Ed0100C02f Gossypium arboreum 7-10 dpa fiber library    -   Gossypium arboreum cDNA clone GA_Ed0100C02f.

EST3 Complementary

-   -   gi|18099677|gb|BM358931.1|BM358931    -   GA_Ea0014H01r Gossypium arboreum 7-10 dpa fiber library    -   Gossypium arboreum cDNA clone GA_Ea0014H01r.

From a contig analysis, it was determined that EST2 and EST3 are overlapESTs, designated EST2-3, as shown in FIG. 15. The predicted amino acidsequences of the two contigs, EST1 and EST2-3, as well as of EST2 and ofEST3, are shown in FIG. 16. An alignment of the predicted amino acidsequences of the two contigs, EST2 and EST2_(—)3, with that of theSterculia CPA-FAS is shown in FIG. 17.

The prevalence of sequencing errors in EST sequences frequently invitesan additional analysis of predicted amino acid sequences encoded byESTs. In some instances, this is due to the fact that errors insequencing result in frameshift, which result in non-coherent amino acidsequences; in other words, a direct translation of the encoded sequencein an EST may not result in a full length polypeptide fragment. Thus, apredicted amino acid sequence can be aligned with a known sequence, suchthat errors can be observed; these errors are corrected, and a coherentamino acid sequence reconstructed. An analysis of the three cotton ESTsrevealed that EST3 contained several apparent sequencing errors. Bycomparing the encoded sequence with the analogous region of theSterculia CPA-FAS amino acid sequence, two apparent reading frames wereobserved, reading frame+2 and reading frame+3, as follows:

Combining the two reading frames result in the amino acid sequencebelow; this sequence (SEQ ID NO:9) comprises the highlighted portions ofreading frames +2 and +3 above, and is shown in FIG. 16.

Combined reading frames:

Characterization of Cotton CPA-FAS

Western Blot analysis with Antibody to Sterculia CPA-FAS

Antibody to Sterculia CPA-FAS, prepared as described in Example 4, wasused to stain protein extracts obtained from cotton and Sterculiaprotein extracts by the following procedure. Sterculia was grown asdescribed previously, and embryo tissue extracted. Cotton plants(Gossypium hirsutum L.) were grown in a growth room with 14-hour lightand 10 hour dark cycle. Root, stem and leaf tissues were harvested fromthese plants two weeks after germination. Embryo tissues were harvestedfrom cotton plants grown in a greenhouse under the natural light andtemperature conditions about 10 days after flowering. The same types oftissues were used for both Western Blot and fatty acid analyses.

Tissue samples were obtained from cotton embryos, leaves, stems, androots, and from Sterculia embryos. Protein extracts were obtained byhomogenizing 0.5 gm of tissue in 2 ml ice-cold buffer (20 mM Tris-HCl,pH7.5, 0.2 M NaCl, 10 mM β-mercaptoethanol). The concentration of theprotein in the homogenate was determined, and then 150 :g of cottontissue proteins and 20 :g of proteins from Sterculia embryos were loadedonto 8% SDS polyacrylaminde gel. After electrophoresis, the separatedproteins were transferred to a nitrocellulose membrane for Western Blotanalysis, with the antibody against Sterculia CPA-FAS.

The results of the Western Blot analysis are shown in FIG. 18, where aprotein in the cotton tissue extracts binds the Sterculia antibody;based upon its antibody binding, and the presence of the EST sequencesin cotton tissues, this protein was identified as a cotton CPA-FAS. Theresults demonstrate that antibody against Sterculia CPA-FAS cross-reactswith the cotton CPA-FAS extremely well, indicating that the two proteinsare very similar. Moreover, the size of cotton CPA-FAS is almost thesame as its counterpart in Sterculia. In cotton, CPA-FAS is highlyexpressed in young embryo, stem, and root tissues, but not in leaftissues. These observations are in general agreement with the productionof carbocyclic fatty acids in these tissues, as described below.

Fatty Acid Analysis

Fatty acids were analyzed from embryo, leaf, stem, and root tissues fromcotton, and the results shown below.

Total cyclopropene fatty acids, which include 18:1c+19:1c: Root   34%Stem 27.5% Leaf 0.00% Embryo  0.3%

Total cyclopropane fatty acids, which include 19:0c: Root 0.88% Stem0.48% Leaf 0.00% Embryo 0.23%

TABLE 1 Fatty acid analysis of Cotton Tissues Fatty Acid Root Stem LeafEmbryo 16:0 25.9 22.1 21.0 28.4 16:1 0 0 0 0.5 18:1c 25.1 18.3 0 0.318:0 0.5 0.5 2.9 1.6 18:1 2.9 7.0 14.7 12.2 18:2 21.5 27.5 39.5 56.719:1c 8.9 9.2 0 0 19:0c 0.9 0.5 0 0.2 18:3 14.3 15.0 21.92 0.1

These results demonstrate that in cotton root and stem tissues,cyclopropane and cyclopropene fatty acids compose about 30% and about35% of total fatty acids, respectively. But these fatty acids composeonly about 2% of the total fatty acids in more mature embryo tissues,and none in leaf tissues. Of these fatty acids in root and stem tissues,malvalic acid is the most abundant, accounting for about 25% and about20% of total fatty acids in root and stem tissues, respectively.

Thus, in cotton tissues, the abundance of CPA-FAS in different tissues,as demonstrated by Western Blot analysis as described above, isgenerally in agreement with the percentage of cyclic fatty acid. Itshould be noted that in plants in which cyclic fatty acids have beenobserved, cyclopropane and cyclopropene fatty acids are synthesized veryearly in seed development, where they can initially represent arelatively high proportion of the total fatty acids; however, thesynthesis of these fatty acids decreases as the seeds mature, so thatthe proportion of these fatty acids decreases to a much lower level inmature seed tissue.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in material science,chemistry, and molecular biology or related fields are intended to bewithin the scope of the following claims.

1. A composition comprising an isolated nucleic acid sequence encoding aplant cyclopropane fatty acid synthase, wherein said sequence is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO 1, 3, 4, 5, and 6 and wherein said sequence encodes a proteinhaving cyclopropane fatty acid synthase activity.
 2. The composition ofclaim 1, wherein the plant is from the order Malvales.
 3. Thecomposition of claim 2, wherein the plant is a Sterculia plant or acotton plant.
 4. The composition of claim 3, wherein the Sterculia plantis a Sterculia foetida plant or the cotton plant is a Gossypium arboreumplant.
 5. The composition of claim 4, wherein the nucleic acid sequencecomprises a sequence selected from the group consisting of SEQ ID NO: 1,3, 4, 5, and
 6. 6. A composition comprising a nucleic acid sequenceencoding a plant cyclopropane fatty acid synthase, wherein said sequenceis at least 90% identical to a sequence selected from the groupconsisting of SEQ ID NO 1, 3, 4, 5, and 6 and wherein said sequenceencodes a protein having cyclopropane fatty acid synthase activityoperably linked to a heterologous promoter.
 7. A composition comprisinga vector comprising a nucleic acid sequence encoding a plantcyclopropane fatty acid synthase, wherein said sequence is at least 90%identical to a sequence selected from the group consisting of SEQ ID NO1, 3, 4, 5, and 6 and wherein said sequence encodes a protein havingcyclopropane fatty acid synthase activity.
 8. A composition comprising apurified polypeptide encoded by a nucleic acid sequence of encoding aplant cyclopropane fatty acid synthase, wherein said sequence is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO 1, 3, 4, 5, and 6 and wherein said sequence encodes a proteinhaving cyclopropane fatty acid synthase activity.
 9. A compositioncomprising a purified plant cyclopropane fatty acid synthase, whereinsaid cyclopropane fatty acid synthase comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:2, 7, 8, 9, 10, and 11and sequences at least 90% identical thereto.
 10. The composition ofclaim 9, wherein the plant is of the order Malvales.
 11. The compositionof claim 10, wherein the plant is a Sterculia plant or a cotton plant.12. The composition of claim 11, wherein the Sterculia plant isSterculia foetida or the cotton plant is a Gossypium arboreum plant. 13.The composition of claim 12, wherein the purified cyclopropane fattyacid synthase comprises amino acid sequence SEQ ID NO:2, or comprises atleast one of amino acid sequences SEQ ID NOs: 7-10, or comprises SEQ IDNO:11.
 14. A composition comprising an isolated nucleic acid sequenceencoding a cyclopropane fatty acid synthase of any of claim
 9. 15. Anorganism transformed with a heterologous gene encoding a plantcyclopropane fatty acid synthase, wherein the gene comprises a nucleicacid sequence of claim 1, or a nucleic acid sequence which encodes aplant cyclopropane fatty acid synthase of claim
 9. 16. A planttransformed with a heterologous gene encoding a plant cyclopropane fattyacid synthase, wherein the gene comprises a nucleic acid sequence ofclaim 1, or a nucleic acid sequence which encodes a plant cyclopropanefatty acid synthase of claim
 9. 17. A plant cell transformed with aheterologous gene encoding a plant cyclopropane fatty acid synthase,wherein the gene comprises a nucleic acid sequence of claim 1, or anucleic acid sequence which encodes a plant cyclopropane fatty acidsynthase of any of claim
 9. 18. A plant seed transformed with aheterologous gene encoding a plant cyclopropane fatty acid synthase,wherein the gene comprises a nucleic acid sequence of claim 1, or anucleic acid sequence which encodes a plant cyclopropane fatty acidsynthase of any of claim
 9. 19. Oil from a transgenic plant of claim 18.20. A bacteria transformed with a heterologous gene encoding a plantcyclopropane fatty acid synthase, wherein the gene comprises a nucleicacid sequence of claim 1, or a nucleic acid sequence which encodes aplant cyclopropane fatty acid synthase of any of claim
 9. 21. Oil fromthe transgenic bacterial of claim
 20. 22. A composition comprising anisolated nucleic acid sequence encoding a protein comprising an aminoacid sequence homologous to an amino terminus of a plant cyclopropanefatty acid synthase.
 23. A composition comprising a purified proteincomprising an amino acid sequence homologous to an amino terminus of aplant cyclopropane fatty acid synthase.
 24. The composition of claim 23,wherein the amino terminus comprises approximately the first about 420to about 470 amino acids of a plant cyclopropane fatty acid synthase.25. The composition of claim 24, wherein the amino terminus comprisesthe first about 420 to 470 amino acids of the amino terminus of SEQ IDNO:2 or comprises amino acid SEQ ID NO:7.
 26. A method for expressing aplant cyclopropane fatty acid synthase in a plant, comprising; a)providing i) a vector comprising a nucleic acid sequence which encodes aplant CPA-FAS or portion thereof, and ii) plant tissue; and b)transfecting the plant tissue with the vector under conditions such thatthe synthase is expressed.
 27. A method for decreasing expression ofCPA-FAS in plants, comprising: a) providing i) a vector comprising anucleic acid sequence encoding an antisense sequence corresponding to anucleic acid sequence which encode a plant CPA-FAS or portion thereof,and i) plant tissue; and b) transfecting the plant tissue with thevector under conditions such that the antisense sequence is expressedand the expression of CPA-FAS is decreased.
 28. A method for decreasingexpression of CPA-FAS in plants, comprising: a) providing i) vectorencoding an siRNA targeted to a nucleic acid sequence which encodes aplant CPA-FAS or portion thereof, and ii) plant tissue; and b)transfecting the plant tissue with the vector under conditions such thatthe siRNA is expressed and the expression of CPA-FAS is decreased.